Androgen-regulated PMEPA1 gene and polypeptides

ABSTRACT

This invention relates to the androgen-regulated gene, PMEPA1, and proteins encoded by this gene, including variants and analogs thereof. Also provided are other androgen-regulated nucleic acids, a polynucleotide array containing these androgen-regulated nucleic acids, and methods of using the polynucleotide array in the diagnosis and prognosis of prostate cancer.

CROSS REFERENCE TO RELATED APPLICATIONS

The present application is a continuation-in-part of copending U.S.application Ser. No. 10/390,045, filed Mar. 18, 2003, which is adivisional of U.S. Applicaton Ser. No. 09/769,482, filed Jan. 26, 2001,allowed, which is based upon U.S. provisional applications S. No.60/178,772, and 60/179,045, filed Jan. 28, 2000, and Jan. 31, 2000,respectively, priority to which is claimed under 35 U.S.C. § 119(e). Theentire disclosures of these applications are expressly incorporatedherein by reference.

GOVERNMENT INTEREST

The invention described herein may be manufactured, licensed, and usedfor governmental purposes without payment of royalties to us thereon.

FIELD OF THE INVENTION

The present invention relates to tumor suppressor genes, and inparticular, PMEPA1 genes, and the proteins encoded by these genes,including variants and/or analogs thereof. More particularly, thepresent invention is based in part on the discovery that PMEPA1polypeptides inhibit cancer cell growth. The present invention alsorelates to novel, androgen-regulated nucleic acids, polynucleotidearrays containing androgen-regulated nucleic acids, such as PMEPA1, andmethods of using the array in the evaluation of hormone-related cancers,such as prostate cancer.

BACKGROUND

Prostate cancer (CaP) is the most common malignancy in American men andsecond leading cause of cancer mortality (1). Serum-prostate specificantigen (PSA) tests have revolutionized the early detection of CaP (2).Although PSA has revolutionized early detection of prostate cancer,there is still a very high false positive rate. The increasing incidenceof CaP has translated into wider use of radical prostatectomy as well asother therapies for localized disease (3-5). The wide spectrum ofbiologic behavior (6) exhibited by prostatic neoplasms poses a difficultproblem in predicting the clinical course for the individual patient(3-5). Traditional prognostic markers such as grade, clinical stage, andpretreatment PSA have limited prognostic value for individual men (3-5).A more reliable technique for the evaluation and prognosis of CaP isdesirable.

Molecular studies have shown a significant heterogeneity betweenmultiple cancer foci present in a cancerous prostate gland (7, 8). Thesestudies have also documented that the metastatic lesion can arise fromcancer foci other than those present in dominant tumors (7).Approximately 50-60% of patients treated with radical prostatectomy forlocalized prostate carcinomas are found to have microscopic disease thatis not organ-confined, and a significant portion of these patientsrelapse (9). Therefore, identification and characterization of geneticalterations defining CaP onset and progression is crucial inunderstanding the biology and clinical course of the disease.

Despite recent intensive research investigations, much remains to belearned about specific molecular defects associated with CaP onset andprogression (6, 10-15). Alterations of the tumor suppressor gene p53,bcl-2 and the androgen receptor (AR), are frequently reported inadvanced CaP (6, 10-15). However, the exact role of these geneticdefects in the genesis and progression of CaP is poorly understood (6,10-15). Recent studies have shown that the “focal p53 immunostaining” orbcl-2 immunostaining in radical prostatectomy specimens were independentprognostic markers for cancer recurrence after surgery (16-19).Furthermore, the combination of p53 and bcl-2 alterations was a strongerpredictor of cancer recurrence after radical prostatectomy (18).

The roles of several new chromosome loci harboring putativeproto-oncogenes or tumor suppressor genes are being currently evaluatedin CaP (7-13). High frequency of allelic losses on 8p21-22, 7q31.1,10q23-25 and 16q24 loci have been shown in CaP (6, 10-15). PTEN1/MMAC1,a recently discovered tumor suppressor gene on chromosome 10q25, isfrequently altered in advanced CaP (20, 21). Gains of chromosome 8q24harboring c-myc and prostate stem-cell antigen (PSCA) genes have alsobeen shown in prostate cancer (22, 23). Studies utilizing comparativegenomic hybridization (CGH) have shown frequent losses of novelchromosomal loci including 2q, 5q and 6q and gains of 11p, 12q, 3q, 4qand 2p in CaP (24, 25). The inventors have recently mapped a 1.5megabase interval at 6q16-21 which may contain the putative tumorsuppressor gene involved in a subset of prostate tumors. The risk for 6qLOH to non-organ confined disease was five fold higher than for organconfined disease (26). Chromosome regions, 1q24-25 and Xq27-28 have beenlinked to familial CaP (27, 28).

It is evident that multiple molecular approaches need to be explored toidentify CaP-associated genetic alterations. Emerging strategies fordefining cancer specific genetic alterations and characterizing androgenregulated genes in rat prostate and LNCaP human prostate cancer cellmodels include, among others, the study of global gene expressionprofiles in cancer cells and corresponding normal cells by differentialdisplay (DD) (29) and more recent techniques, such as serialamplification of gene expression (SAGE) (30) and DNA micro-arrays (31;U.S. Pat. Nos. 5,744,305 and 5,837,832 which are herein incorporated byreference) followed by targeted analyses of promising candidates. Ourlaboratory has also employed DD, SAGE and DNA microarrays to study CaPassociated gene expression alterations (32-33). Each of thesetechniques, however, is limited. The number of transcripts that can beanalyzed is the major limitation encountered in subtractivehybridization and differential display approaches. Furthermore, whilecDNA microarray approaches can determine expression of a large number ofgenes in a high throughput manner, the current limitations of cDNAarrays include the presence of specific arrays used for analyses and theinability to discover novel genes.

While alterations of critical tumor-suppressor genes and oncogenes areimportant in prostate tumorogenesis, it is also recognized that hormonalmechanisms play equally important roles in prostate tumorogenesis. Thecornerstone of therapy in patients with metastatic disease is androgenablation, commonly referred to as “hormonal therapy (34),” which isdependent on the inhibition of androgen signaling in prostate cancercells. Androgen ablation can be achieved, for example, by orchiectomy,by the administration of estrogen, or more recently by one of theluteinizing hormone-releasing hormone agonists. Recent clinical trialshave demonstrated the efficacy of combining an antiandrogen toorchiectomy or a luteinizing hormone-releasing hormone to block theremaining androgens produced by the adrenal glands. Althoughapproximately 80% of patients initially respond to hormonal ablation,the vast majority of patients eventually relapse (35), presumably due toneoplastic clones of cells which become refractory to this therapy.

Alterations of the androgen receptor gene by mutations in the hormonebinding domain of the AR or by amplification of the AR gene have beenreported in advanced stages of CaP. Much remains to be learned, however,about the molecular mechanisms of the AR-mediated cell signaling inprostate growth and tumorogenesis (36-43). Our earlier studies have alsodescribed mutations of the AR in a subset of CaP (40). Mutations of theAR are reported to modify the ligand (androgen) binding of the AR bymaking the receptor promiscuous, so that it may bind to estrogen,progesterone, and related molecules, in addition to the androgens (36,38, 42). Altered ligand binding specificity of the mutant AR may provideone of the mechanisms for increased function in cancer cells.Amplifications of the AR gene in hormone-refractory CaP represent yetanother scenario where increase in AR function is associated with tumorprogression (44, 45).

Several growth factors commonly involved in cell proliferation andtumorogenesis, e.g., IGF1, EGF, and others, have been shown to activatethe transcription transactivation functions of the AR (46). Theco-activator of the AR transcription factor functions may also play arole in prostate cancer (47). Recent studies analyzing expression of theandrogen-regulated genes (ARGs) in hormone sensitive and refractoryCWR22 nude mice xenograft models (48) have also shown expression ofseveral androgen regulated genes in AR positive recurrent tumorsfollowing castration, suggesting activation of AR in these tumors (49).

In addition to the alterations of the androgen signaling pathway(s) inprostate tumor progression, androgen mechanisms are suspected to play arole in the predisposition to CaP. Prolonged administration of highlevels of testosterone has been shown to induce CaP in rats (50-52).Although recent evidence suggests an association of androgen levels andrisk of CaP, this specific observation remains to be established. (53).An independent line of investigations addressing the length of inheritedpolyglutamine (CAG) repeat sequence in the AR gene and CaP risk haveshown that men with shorter repeats were at high risk of distantmetastasis and fatal CaP (54, 55). Moreover, the size distribution of ARCAG repeats in various ethnic groups has also suggested a possiblerelationship of shorter CAG repeats and increased prostate cancer risksin African-American men (56, 57). Biochemical experiments evaluatingAR-CAG repeat length and in vitro transcription transactivationfunctions of the AR revealed that AR with shorter CAG repeats possesseda more potent transcription transactivation activity (58). Thus,molecular epidemiologic studies and biochemical experimentation suggestthat gain of AR function, consequently resulting in transcriptionaltransactivation of downstream targets of the AR gene, may play animportant role in CaP initiation. However, downstream targets of AR mustbe defined in order to understand the biologic basis of theseobservations.

The biologic effects of androgen on target cells, e.g., prostaticepithelial cell proliferation and differentiation as well as theandrogen ablation-induced cell death, are likely mediated bytranscriptional regulation of ARGs by the androgen receptor (reviewed in59). Abrogation of androgen signaling resulting from structural changesin the androgen gene or functional alterations of AR due to modulationof AR functions by other proteins would have profound effects ontranscriptional regulation of genes regulated by AR and, thus, on thegrowth and development of the prostate gland, including abnormal growthcharacterized by benign prostatic hyperplasia and prostatic cancer. Thenature of ARGs in the context of CaP initiation and progression,however, remains largely unknown. Since forced proliferation of the ARprostate cancer cells lacking AR induces cell-death related phenotypes(60), the studies utilizing AR expression via heterologous promoters incell cultures have failed to address the observations relating to gainof AR functions and prostate cancer progression. Moreover, suitableanimal models to assess gain of AR functions do not exist. Therefore,the expression profile of androgen responsive genes (ARGs) has potentialto serve as read-out of the AR signaling status. Such a read-out mayalso define potential biomarkers for onset and progression of thoseprostate cancers which may involve abrogation of the androgen signalingpathway. Furthermore, functional analysis of androgen regulated geneswill help understand the biochemical components of the androgensignaling pathways.

SUMMARY OF THE INVENTION

The present invention relates to the identification and characterizationof a novel androgen-regulated gene that exhibits abundant expression inprostate tissue. The novel gene has been designated PMEPA1. Our workwith PMEPA1 is further described in U.S. Provisional Application S. No.60/378,949, filed May 10, 2002, and PCT Application No. PCT/US03/XXXXX,filed May 9, 2003, the entire disclosures of which are herebyincorporated by reference.

The invention provides the isolated nucleotide sequence of PMEPA1 orfragments thereof and nucleic acid sequences that hybridize to PMEPA1.These sequences have utility, for example, as markers of prostate cancerand other prostate-related diseases, and as targets for therapeuticintervention in prostate cancer and other prostate-related diseases. Theinvention further provides a vector that directs the expression ofPMEPA1, and a host cell transfected or transduced with this vector.

In another embodiment, the invention provides a method of detectingprostate cancer cells in a biological sample, for example, by usingnucleic acid amplification techniques with primers and probes selectedto bind specifically to the PMEPA1 sequence.

In another aspect, the invention relates to an isolated polypeptideencoded by the PMEPA1 gene or a fragment thereof, and antibodiesgenerated against the PMEPA1 polypeptide, peptides, or portions thereof,which can be used to detect, treat, and prevent prostate cancer.

In another aspect, the invention provides variants of the PMEPA1polypeptide that retain at least one of the following abilities:inhibiting cancer cell growth, reducing the expression of an androgenreceptor, or modulating the expression of a gene whose transcription isregulated by the androgen receptor. In one embodiment, these variantsare at least 95% identical to SEQ ID NO:3 and inhibit the growth ofprostate cancer cells (e.g., LNCaP cells), as measured, for example, ina colony-forming assay.

In another aspect, the invention provides a method of inhibiting thegrowth of a cancer cell, comprising administering these variants to thecancer cell in an amount effective to inhibit the growth of the cancercell. In one embodiment the cancer cell is a prostate cancer cell. Thepolypeptide may be administered directly to the cell or indirectly usinga vector containing a polynucleotide sequence that encodes the variant.These methods include therapeutic methods of treating cancer, and inparticular, prostate cancer.

A further embodiment of the invention provides a method of reducing theexpression of an androgen receptor or modulating the expression of genesthat are transcriptionally regulated by androgen receptor, including,but not limited to the prostate-specific antigen (PSA) gene, the PSMAgene, and the PCGEM1 gene. Thus, in one aspect, the invention provides amethod of reducing the expression in a cancer cell of an androgenreceptor or modulating (i.e., increasing or decreasing) the expressionof a gene whose transcription is regulated by the androgen receptor,comprising administering the variants described above to the cancercell, in an amount effective to reduce the androgen receptor or modulatethe expression of the gene in the cancer cell. In one embodiment thecancer cell is a prostate cancer cell. The polypeptide may beadministered directly to the cell or indirectly using a vectorcontaining a polynucleotide sequence that encodes the variant.

In yet another aspect, the invention provides variants of the PMEPA1polypeptide having at least one mutation and/or deletion in the at leastone of the PY motifs of PMEPA1, as discussed in further detail below.Such mutations reduce the cell growth inhibitory effects of PMEPA1.These PMEPA1 variants can be used, for example, to define cellularproteins through which PMEPA1 interacts, directly or indirectly, tomediate cell growth inhibitory functions.

In a still further embodiment, the invention provides thepolynucleotides that encode the PMEPA1 variants, as well as methods (asdescribed above for a polypeptide comprising SEQ ID NO:3) of using thesevariants, for example, to inhibit cancer cell growth, including prostatecancer, and/or to reduce the expression of an androgen receptor and/orto modulate the expression of a gene whose transcription is regulated bythe androgen receptor.

The present invention also relates to a polynucleotide array comprising(a) a planar, non-porous solid support having at least a first surface;and (b) a first set of polynucleotide probes attached to the firstsurface of the solid support, where the first set of polynucleotideprobes comprises polynucleotide sequences derived from genes that areup-regulated, such as PMEPA1, or down-regulated in response to androgen,including genes downstream of the androgen receptor gene and genesupstream of the androgen receptor gene that modulate androgen receptorfunction. In another embodiment of the invention the polynucleotidesimmobilized on the solid support include genes that are known to beinvolved in testosterone biosynthesis and metabolism. In anotherembodiment of the invention the oligonucleotides immobilized on thesolid support include genes whose expression is altered in prostatecancer or is specific to prostate tissue.

In another embodiment, the invention provides a method for the diagnosisor prognosis of prostate cancer, comprising (a) hybridizing nucleicacids of a target cell of a patient with a polynucleotide array, asdescribed above, to obtain a first hybridization pattern, where thefirst hybridization pattern represents an expression profile ofandrogen-regulated genes in the target cell; (b) comparing the firsthybridization pattern of the target cell to a second hybridizationpattern, where the second hybridization pattern represents an expressionprofile of androgen-regulated genes in prostate cancer, and (c)diagnosing or prognosing prostate cancer in the patient.

Thus, a first aspect of the present invention is directed towards amethod for analysis of radical prostatectomy specimens for theexpression profile of those genes involved in androgen receptor-mediatedsignaling. In a preferred embodiment, computer models may be developedfor the analysis of expression profiles. Another aspect of the inventionis directed towards a method of correlating expression profiles withclinico-pathologic features. In a preferred embodiment, computer modelsto identify gene expression features associated with tumor phenotypesmay be developed. Another aspect of the invention is directed towards amethod of distinguishing indolent prostate cancers from those with amore aggressive phenotype. In a preferred embodiment, computer models tosuch cancers may be developed. Another aspect of the invention isdirected towards a method of analyzing tumor specimens of patientstreated by radical prostate surgery to help define prognosis. Anotheraspect of the invention is directed towards a method of screeningcandidate genes for the development of a blood test for improvedprostate cancer detection. Another aspect of the invention is directedtowards a method of identifying androgen regulated genes that may serveas biomarkers for response to treatment to screen drugs for thetreatment of advanced prostate cancer.

This invention is further directed to a method of identifying anexpression profile of androgen-regulated genes in a target cell,comprising hybridizing the nucleic acids of the target cell with apolynucleotide array, as described above, to obtain a hybridizationpattern, where the hybridization pattern represents the expressionprofile of androgen-regulated genes in the target cell.

Additional features and advantages of the invention will be set forth inthe description which follows, and in part will be apparent from thedescription, or may be learned by practice of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a Northern blot showing that PMEPA1 is expressed at highlevels in prostate tissue. Multiple tissue northern blots werehybridized with PMEPA1 and GAPDH probes. The arrows indicate the twovariants of the PMEPA1 transcript.

FIG. 2 shows the androgen-dependent expression of PMEPA1. FIG. 2A is aNorthern blot using PMEPA1 probe with mRNA derived from LNCaP cells withor without R1881 treatment for various durations. FIG. 2B is a Northernblot of PMEPA1 expression in primary epithelial cell cultures of normalprostate and prostate and breast cancer cell lines.

FIGS. 3A-H show the effect of PMEPA1 on colony formation. Prostate tumorcell lines: C4 (FIG. 3A), C4-2 (FIG. 3B), C4-2B (FIG. 3C), LNCaP (FIG.3D), DU145 (FIG. 3E), and PC3 (FIG. 3F) were transfected with 3 μg ofeach of PMEPA1-V5-pcDNA3.1 (PMEPA1) and pcDNA3.1 vector (Vector) intriplicate sets. In a separate experiments LNCaP (FIG. 3G) and PC3 (FIG.3H) cells were transfected with control vector or expression vectorsencoding wt-PMEPA1 or PMEPA1-PY mutants (1. PMEPA1-V5-pcDNA3.1, 2.PMEPA1-PY1m-pcDNA3.1, 3. PMEPA1-PY2m-pcDNA3.1, 4. PMEPA1-PY1m/PY2m-pcDNA3.1, and 5. pcDNA3.1). Transfected cells were selected forplasmid-containing cells with G418 for 3 weeks and surviving cells werefixed and stained with crystal violet. In each experiment, the number ofcolonies per dish were counted and displayed as histograms, representingthe mean number of colonies±SD of the triplicate sets. For each cellline, a photograph of one dish of cells treated with 3 μg of eachplasmid is also shown.

FIG. 4A shows PMEPA1-mediated down regulation of androgen receptor andits functional consequences on androgen receptor regulated genes. LNCaPcells stably transfected with PMEPA1-GFP and pEGFP (control) plasmidswere cultured in medium with cFBS for 5 days and then were stimulatedwith R1881 at 0.1 nM. Cells were harvested for Western blotting at 0 h,12 h and 24 h after androgen stimulation. Antibodies against androgenreceptor, PSA, PSMA and tubulin were used to detect correspondingproteins on Western Blots.

FIG. 4B shows that PMEPA1 does not reduce androgen receptor expressionthrough a non-specific, PMEPA1-induced effect on theubiquitin-proteasome pathway. Stable PMEPA1-GFP-Tet-LNCaP transfectants(Tet-off system) were cultured in proper medium with or withouttetracycline for 10 days and were applied for immunoblotting. Antibodiesagainst androgen receptor, GFP, p27 and tubulin were used to detect thecorresponding proteins.

FIG. 5 shows the effect of PMEPA1 on cell proliferation. StablePMEPA1-GFP-Tet-LNCaP transfectants were seeded in 96-well plates with orwithout 1 μg/ml of tetracycline in the medium. The cell proliferationwas measured using the CellTiter 96 Aqueous One Solution kit at theindicated time. Tet+ and Tet− denote the cell culture medium with orwithout tetracycline, respectively. The OD values reflecting the cellnumbers are significantly different (p<0.01) between the two groupsexcept on day one.

FIG. 6 defines binding of PMEPA1 to NEDD4 proteins. The in vitrotranscription/translation products ([³⁵S]Methionine-labeled lysates)derived from expression plasmids: PMEPA1-V5-pcDNA3.1 (Lanes 1, 5),PMEPA1-PY1m-pcDNA3.1 (Lanes 2, 6), PMEPA1-PY2m-pcDNA3.1 (Lanes 3, 7),and PMEPA1-PY1m/PY2m-pcDNA3.1 (Lanes 4, 8) were incubated withGST-NEDD4-WW-Sepharose beads (Lanes 1-4) or control GST beads (Lanes5-8) and [³⁵S] Methionine labeled proteins bound toGST-NEDD4-WW-Sepharose beads were solublized in sample buffer and wereresolved by SDS-PAGE gel. Equal amounts of [³⁵S]Methionine lysatescorresponding to samples in lanes 1-4 were run on SDS-PAGE gel withoutGST pull-down (Lane 9-12).

FIG. 7 represents an immunoprecipitation assay. 293 cells wereco-transfected with expression vectors encoding NEDD4-GFP and one offollowing fusion proteins: PMEPA1-V5 (Lane 1), PMEPA1-PY1m-V5 (Lane 2),PMEPA1-PY2m-V5 (Lane 3) or PMEPA1-PY1m/PY2m-V5 (Lane 4). The celllysates from each group were immunoprecipitated with anti-GFP antibodythen subjected to immunoblotting (blot a). Cell lysates from each groupwithout immunoprecipitation were also processed for immunoblotting(blots b and c) to serve as a control. Blots a and b were detected byanti-V5 antibody and blot c was detected by anti-GFP antibody.

FIG. 8 shows PMEPA1 expression in CWR22 xenograft tumors. Lane 1, samplefrom CWR22 tumor (androgen dependent). Lanes 2-5, samples from 4individual CWR22R tumors (AR positive but androgen independent).

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a method useful in the diagnosis andprognosis of prostate cancer. An aspect of the invention provides amethod to identify ARGs, such as PMEPA1, that exhibit stabletranscriptional induction/repression in response to androgen and havepotential as surrogate markers of the status of the androgen signalingin normal and cancerous epithelial cells of prostate.

A second aspect of the invention provides for use of the expressionprofiles resulting from these methods in diagnostic methods, including,but not limited to, characterizing the treatment response to “hormonaltherapy,” correlating expression profiles with clinico-pathologicfeatures, distinguishing indolent prostate cancers from those with amore aggressive phenotype, analyzing tumor specimens of patients treatedby radical prostate surgery to help define prognosis, screeningcandidate genes for the development of a polynucleotide array for use asa blood test for improved prostate cancer detection, and identifyingandrogen regulated genes that may serve as biomarkers for response totreatment to screen drugs for the treatment of advanced prostate cancer.

As will be readily appreciated by persons having skill in the art, thesegene sequences and ESTs described herein can easily be synthesizeddirectly on a support, or presynthesized polynucleotide probes may beaffixed to a support as described, for example, in U.S. Pat. Nos.5,744,305, 5,837,832, and 5,861,242, each of which is incorporatedherein by reference. Furthermore, such arrays may be made in a widenumber of variations, combining, probes derived from sequencesidentified by the inventors as up-regulated or down-regulated inresponse to androgen and listed in Table 3 (genes and ESTs derived fromthe inventors' SAGE library that are up-regulated and down-regulated byandrogens) with any of the sequences described in Table 4 (candidategenes and ESTs whose expression are potentially prostate specific orrestricted), Table 5 (previously described genes and ESTs, includingthose associated with androgen signaling, prostate specificity, prostatecancer, and nuclear receptors/regulators with potential interaction withandrogen receptors), Table 6 (genes and ESTs identified from the NIHCGAP database that are differentially expressed in prostate cancer),Table 7 (androgen regulated genes and ESTs derived from the CPDR GenomeSystems ARG Database) and Table 8 (other genes associated with cancers).Tables 3-8 are located at the end of the specification at the end of the“Detailed Description” section and before the “References.” In Table 3,genes in bold type are known androgen-regulated genes based on MedlineSearch. In Table 4, genes in bold type are known prostate-specificgenes.

Such arrays may be used to detect specific nucleic acid sequencescontained in a target cell or sample, as described in U.S. Pat. Nos.5,744,305, 5,837,832, and 5,861,242, each of which is incorporatedherein by reference. More specifically, in the present invention, thesearrays may be used in methods for the diagnosis or prognosis of prostatecancer, such as by assessing the expression profiles of genes, derivedfrom biological samples such as blood or tissues, that are up-regulatedand down-regulated in response to androgen or otherwise involved inandrogen receptor-mediated signaling. In a preferred embodiment,computer models may be developed for the analysis of expressionprofiles. Moreover, such polynucleotide arrays are useful in methods toscreen drugs for the treatment of advanced prostate cancer. In thesescreening methods, the polynucleotide arrays are used to analyze howdrugs affect the expression of androgen-regulated genes that areinvolved in prostate cancer.

SAGE analysis. The SAGE technology is based on three main principles: 1)A short sequence tag (10-11 bp) is generated that contains sufficientinformation to identify a transcript, thus, each tag represents asignature sequence of a unique transcript; 2) many transcript tags canbe concatenated into a single molecule and then sequenced, revealing theidentity of multiple tags simultaneously; 3) quantitation of the numberof times a particular tag is observed provides the expression level ofthe corresponding transcript (30). The schematic diagram and the detailsof SAGE procedure can be obtained from the web site:www.genzyme.com/SAGE.

About fifty percent of SAGE tags identified by the inventors representESTs which need to be further analyzed for their protein codingcapacity. The known genes up-regulated or down-regulated by four-fold(p<0.05) were broadly classified on the basis of the biochemicalfunctions. SAGE tag defined ARGs were grouped under followingcategories: transcriptional regulators; RNA processing and translationregulators; protein involved in genomic maintenance and cell cycle;protein trafficking/chaperone proteins; energy metabolism, apoptosis andredox regulators; and signal transducers. As determined by PubMeddatabase searches, a majority of genes listed in Table 3 have not beendescribed as androgen regulated before. This is the first comprehensivelist of the functionally defined genes regulated by androgen in thecontext of prostatic epithelial cells.

Although promising candidate ARGs have been identified using theseapproaches, much remains to be learned about the complete repertoire ofthese genes. SAGE provides both quantitative and high throughputinformation with respect to global gene expression profiles of known aswell as novel transcripts. We have performed SAGE analysis of the ARGsin the widely studied hormone responsive LNCaP prostate cancer cellstreated with and without synthetic androgen, R1881. Of course, this SAGEtechnique could be repeated with hormones other than R1881, includingother synthetic or natural androgens, such as dihydroxytestosterone, topotentially obtain a slightly different ARG expression panel. A goal ofthe inventors was to identify highly induced and repressed ARGs in LNCaPmodel which may define a panel of surrogate markers for the statusandrogen signaling in normal as well as cancerous prostate. Here, wereport identification and analyses of a comprehensive database of SAGEtags corresponding to well-characterized genes, expressed sequence tags(ESTs) without any protein coding information and SAGE tagscorresponding to novel transcripts. This is the first report describinga quantitative evaluation of the global gene expression profiles of theARGs in the context of prostatic cancer cells by SAGE. We have furtherdefined the ARGs on the basis of their known biologic/biochemicalfunctions. Our study provides quantitative information on about 23,000transcripts expressed in LNCaP cells, the most common cell line used inprostate cancer research. Finally, comparison of the LNCaP SAGE taglibrary and 35 SAGE tag libraries representing diverse cell type/tissueshave unraveled a panel of genes whose expression are prostate specificor prostate abundant. Utilizing the LNCaP prostate cancer cells, theonly well-characterized androgen responsive prostatic epithelial cells(normal or cancerous), we have identified a repertoire of androgenregulated genes by SAGE.

Utilizing cell-culture systems and cell-signaling agents or exogenousexpression of p53 and APC genes, SAGE technology has identified novelphysiologically relevant transcriptional target genes which haveunraveled new functions of p53 and APC genes (61-64). Our analysis ofARGs has provided identification and quantitative assessment ofinduction or repression of a global expression profile of ARGs in LNCaPcells. ARGs resulting from the mutational defects of the AR and thoseARGs unaffected by AR mutations may be identified in this model system.Subsequent androgen regulation analysis of the selected ARGs inAR-positive, primary cultures of normal prostatic epithelial cells, andARGs expression analysis in normal and tumor tissues will clarify normalor abnormal regulation of these ARGs. A panel of highlyinducible/repressible ARGs identified by the inventors may providebio-indicators of the AR transcription factor activity in physiologiccontext. These AR Function Bio-indicators (ARFBs) are useful inassessing the risk of CaP onset and/or progression. Moreover,identification or ARGs may also help in defining the therapeutic targetswhich could lead to effective treatment for hormone refractory cancer,currently a frustrating stage of the disease with limited therapeuticoptions.

Characterization of a SAGE-defined EST that exhibited the highest levelof induction in LNCaP cells responding to R1881 led to the discovery ofa novel, androgen-induced gene, PMEPA1, which encodes a polypeptide witha type lb transmembrane domain. A Protein sequence similarity searchshowed homology to C18orf1, a novel gene located on chromosome 18 thatis mainly expressed in brain with multiple transcriptional variants(Yoshikawa et al., 1998). In addition to the sequence similarity, PMEPA™also shares other features with C18orf1, e.g., similar size of thepredicted protein and similar transmembrane domain as the P1 isoform ofC18orf1. Therefore, it is likely that other isoforms of PMEPA1 mayexist.

Database searches showed that the PMEPA1 sequence matched to genomicclones RP5-1059L7 and 718J7 which were mapped to chromosome20q13.2-13.33. Gain of 20q has been observed in many cancer types,including prostate, bladder, melanoma, colon, pancreas and breast(Brothman et al., 1990; Richter et al., 1998; Bastian et al., 1998; Komet al., 1999; Mahlamaki et al., 1997; Tanner et al., 1996). Chromosome20q gain was also observed during immortalization and may harbor genesinvolved in bypassing senescence (Jarrard et al., 1999; Cuthill et al.,1999). A differentially expressed gene in hormone refractory CaP, UEV-1,mapped to 20q13.2 (Stubbs et al., 1999). These observations indicatethat one or several genes on chromosome 20q may be involved in prostateor other cancer progression. Although we did not observe increasedexpression of PMEPA™ in primary prostate tumors, increased PMEPA1expression was noted in recurrent cancers of CWR22 xenograft.

PMEPA1 expression is upregulated by androgens in a time- andconcentration-specific manner in LNCaP cells. This observationunderscores the potential of measuring PMEPA1 expression as one of thesurrogate markers of androgen receptor activity in vivo in theepithelial cells of prostate tissue. Prostate cancer is androgendependent and its growth in prostate is mediated by a network of ARGsthat remains to be fully characterized. Most prostate cancers respond toandrogen withdrawal but relapse after the initial response (Koivisto etal., 1998). The growth of the relapsed tumors is androgen independenteven though tumors are positive for the expression of the AR (Bentel etal., 1996).

One of the hypotheses of how cancer cells survive and grow in the lowandrogen environment is the sensitization or the activation of the ARpathway (Jenster et al., 1999). Studies have shown increased expressionof the ARGs or amplification of AR in androgen independent prostatecancer tissues (Gregory et al., 1998; Lin et al., 1999). We haveobserved that PMEPA1 was expressed in all CWR22R tumors and increasedexpression in three of four compared with CWR22 tumor. Our data supportthe concept that normally AR-dependent pathways remain activated,despite the absence of androgen in androgen-independent prostate cancer.There are only limited studies that have addressed whether ARGs play arole in the transition from androgen dependent tumor to androgenindependent tumors. The high level of expression only in the prostategland indicates that PMEPA1 might have important roles related toprostate cell biology or physiology. On the basis of homology of PMEPA1to C18orf1 it is tempting to suggest that the PMEPA1 may belong tofamily of proteins involved in the binding of calcium and LDL.

ARGs, including PMEPA1, can be used as biomarkers of AR function readoutin the subset of prostate cancers that may involve abrogation ofandrogen signaling. Furthermore, the newly defined ARGs have potentialto identify novel targets in therapy of hormone refractory prostatecancer.

The nucleic acid molecules encompassed in the invention include thefollowing PMEPA1 nucleotide sequence:

-   ATGGCGGAGC TGGAGTTTGT TCAGATCATC ATCATCGTGG TGGTGATGAT 50-   GGTGATGGTG GTGGTGATCA CGTGCCTGCT GAGCCACTAC AAGCTGTCTG 100-   CACGGTCCTT CATCAGCCGG CACAGCCAGG GGCGGAGGAG AGAAGATGCC 150-   CTGTCCTCAG AAGGATGCCT GTGGCCCTCG GAGAGCACAG TGTCAGGCAA 200-   CGGAATCCCA GAGCCGCAGG TCTACGCCCC GCCTCGGCCC ACCGACCGCC 250-   TGGCCGTGCC GCCCTTCGCC CAGCGGGAGC GCTTCCACCG CTTCCAGCCC 300-   ACCTATCCGT ACCTGCAGCA CGAGATCGAC CTGCCACCCA CCATCTCGCT 350-   GTCAGACGGG GAGGAGCCCC CACCCTACCA GGGCCCCTGC ACCCTCCAGC 400-   TTCGGGACCC CGAGCAGCAG CTGGAACTGA ACCGGGAGTC GGTGCGCGCA 450-   CCCCCAAACA GAACCATCTT CGACAGTGAC CTGATGGATA GTGCCAGGCT 500-   GGGCGGCCCC TGCCCCCCCA GCAGTAACTC GGGCATCAGC GCCACGTGCT 550-   ACGGCAGCGG CGGGCGCATG GAGGGGCCGC CGCCCACCTA CAGCGAGGTC 600-   ATCGGCCACT ACCCGGGGTC CTCCTTCCAG CACCAGCAGA GCAGTGGGCC 650-   GCCCTCCTTG CTGGAGGGGA CCCGGCTCCA CCACACACAC ATCGCGCCCC 700-   TAGAGAGCGC AGCCATCTGG AGCAAAGAGA AGGATAAACA GAAAGGACAC 750-   CCTCTCTAG (SEQ ID NO. 2) 759

The amino acid sequences of the polypeptides encoded by the PMEPA1nucleotide sequences of the invention include:

-   MAELEFVQII IIVVVMMVMV VVITCLLSHY KLSARSFISR HSQGRRREDA 50-   LSSEGCLWPS ESTVSGNGIP EPQVYAPPRP TDRLAVPPFA QRERFHRFQP 100-   TYPYLQHEID LPPTISLSDG EEPPPYQGPC TLQLRDPEQQ LELNRESVRA 150-   PPNRTIFDSD LMDSARLGGP CPPSSNSGIS ATCYGSGGRM EGPPPTYSEV 200-   IGHYPGSSFQ HQQSSGPPSL LEGTRLHHTH IAPLESAAIW SKEKDKQKGH 250-   PL* (SEQ ID NO. 3) 252

The discovery of the nucleic acids of the invention enables theconstruction of expression vectors comprising nucleic acid sequencesencoding polypeptides; host cells transfected or transformed with theexpression vectors; isolated and purified biologically activepolypeptides and fragments thereof; the use of the nucleic acids oroligonucleotides thereof as probes to identify nucleic acid encodingproteins having PMEPA I-like activity; the use of single-stranded senseor antisense oligonucleotides from the nucleic acids to inhibitexpression of polynucleotides encoded by the PMEPA1 gene; the use ofsuch polypeptides and fragments thereof to generate antibodies; the useof the antibodies to purify PMEPA1 polypeptides; and the use of thenucleic acids, polypeptides, and antibodies of the invention to detect,prevent, and treat prostate cancer (e.g., prostatic intraepithelialneoplasia (PIN), adenocarcinomas, nodular hyperplasia, and large ductcarcinomas) and prostate-related diseases (e.g., benign prostatichyperplasia).

As summarized below and explained in further detail in the Examples thatfollow, our evaluation of PMEPA1 indicates it is a prostate-abundantandrogen regulated gene with roles in cell growth control andtumorigenesis. Loss or reduced PMEPA1 expression in prostate cancercorrelates with a higher risk or probability of prostate tumorigenesisor progression (e.g., advanced stages of prostate cancer, such asnon-organ defined cancer, where tumors extend beyond the prostategland), particularly after surgery as primary therapy. Thus, alterationsin the level, expression, and activity of PMEPA1 and/or its encodedpolypeptide provides useful information about the clinical behavior ofprostate cancer. Part of our evaluation involved a PMEPA1 proteinsequence homology search that showed 83% identity to a recently reportedgene, N4WBP4 (Example 8). N4WBP4 encodes a NEDD4 WW domain bindingprotein with two PY motifs that is expressed in mouse embryo [Jolliffeet al., Biochem. J., 351: 557-565, 2000]. The PY motif is a proline-richpeptide sequence with a consensus PPXY sequence (where X can be anyamino acid) that can bind to proteins with WW domains [Jolliffe et al.,Biochem. J., 351: 557-565, 2000; Harvey K et al., Trends Cell Biol., 9:166-169, 1999; Hicke L, Cell, 106: 527-530, 2001; Kumar et al., Biochem.Biophys. Res. Commun., 185: 1155-1161, 1992; Kumar et al., Genomics, 40:435-443, 1997; Sudol M, Trends Biochem. Sci., 21: 161-163, 1996; Harveyet al., J. Biol. Chem., 277: 9307-9317, 2002; and Brunschwig et al.,Cancer Res., 63: 1568-1575, 2003]. NEDD4 was originally identified as adevelopmentally regulated gene in mice and is a ubiquitin-protein ligase(E3) that is involved in the ubiquitin-dependent proteasome-mediatedprotein degradation pathway. Further studies revealed that NEDD4 isimplicated in diverse cellular functions, such as regulation of membranechannels and permeases, endocytosis, virus budding, cell cycle,transcription and protein trafficking [Harvey et al., Trends Cell Biol.,9: 166-169, 1999; Hicke L, Cell, 106: 527-530, 2001]. The WW domainpresent in the NEDD4 protein is a module with two highly conservedtryptophans that bind to several target proteins containing a PY motif.

As explained in Example 9, we discovered that PMEPA1 is a NEDD4 bindingprotein and that the binding of PMEPA1 to NEDD4 is mediated by the PYmotifs of PMEPA1. Mutating the PY motifs significantly reduces thebinding of PMEPA1 to NEDD4. In addition, the homology of PMEPA1 to theNEDD4-binding protein indicates that PMEPA1 may also regulate proteinturnover via ubiquitinylation and proteasome pathways in the cell. Thisis further supported by our observation that PMEPA1 localizes to theGolgi apparatus (Example 11).

Further, we recently found that PMEPA1 expression in LNCaP cells downregulates androgen receptor protein and modulates the expression ofgenes that are transcriptionally regulated by androgen receptor (Example10). This shows that PMEPA1 functions in androgen receptor regulation.

Our data also show that PMEPA1 inhibits the growth of prostate cancercells (Example 12). More specifically, the coding region of PMEPA1 wasinserted into an expression vector and transfected into 293 cell(kidney) and LNCaP cells (prostate cancer). Cell proliferation and cellcycle analysis showed that there was no difference between PMEPA1overexpressed 293 cell and control vector transfected 293 cells. HoweverLNCaP cells overexpressing PMEPA1 exhibited significant cell growthinhibition. Similar growth inhibition was observed in other prostatecancer cell lines.

In addition, in a quantitative evaluation of PMEPA1 expression inprimary prostate cancers, we found that 40 of 62 (64.5%) matchedprostate specimens exhibited decreased expression of PMEPA1 in tumortissues, indicating a correlation between reduced PMEPA1 expression andprostate tumorigenesis (Example 13). When these expression patterns werestratified by organ confined and non-organ confined tumors, a higherpercentage of patients exhibited reduced expression of PMEPA1 innon-organ confined tumor (68%) vs. organ-confined tumor (44%),indicating that reduced PMEPA1 expression correlates with an increasedprobability of advanced prostate cancer.

Nucleic Acid Molecules

In a particular embodiment, the invention relates to certain isolatednucleotide sequences that are free from contaminating endogenousmaterial. A “nucleotide sequence” refers to a polynucleotide molecule inthe form of a separate fragment or as a component of a larger nucleicacid construct. The nucleic acid molecule has been derived from DNA orRNA isolated at least once in substantially pure form and in a quantityor concentration enabling identification, manipulation, and recovery ofits component nucleotide sequences by standard biochemical methods (suchas those outlined in (Sambrook et al., Molecular Cloning: A LaboratoryManual, 3rd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.(www.molecularcloning.com). Such sequences are preferably providedand/or constructed in the form of an open reading frame uninterrupted byinternal non-translated sequences, or introns, that are typicallypresent in eukaryotic genes. Sequences of non-translated DNA can bepresent 5′ or 3′ from an open reading frame, where the same do notinterfere with manipulation or expression of the coding region.

Nucleic acid molecules of the invention include DNA in bothsingle-stranded and double-stranded form, as well as the RNA complementthereof. DNA includes, for example, cDNA, genomic DNA, chemicallysynthesized DNA, DNA amplified by PCR, and combinations thereof. GenomicDNA may be isolated by conventional techniques, e.g., using the SEQ IDNO: 1 or SEQ ID NO:2, or a suitable fragment thereof, as a probe.

The DNA molecules of the invention include full length genes as well aspolynucleotides and fragments thereof. The full length gene may alsoinclude the N-terminal signal peptide. Other embodiments include DNAencoding a soluble form, e.g., encoding the extracellular domain of theprotein, either with or without the signal peptide.

The nucleic acids of the invention are preferentially derived from humansources, but the invention includes those derived from non-humanspecies, as well.

Preferred Sequences

The particularly preferred nucleotide sequence of the invention is SEQID NO:2, as set forth above. The sequence of amino acids encoded by theDNA of SEQ ID NO:2 is shown in SEQ ID NO:3.

Additional Sequences

Due to the known degeneracy of the genetic code, where more than onecodon can encode the same amino acid, a DNA sequence can vary from thatshown in SEQ ID NO:2, and still encode a polypeptide having the aminoacid sequence of SEQ ID NO:3. Such variant DNA sequences can result fromsilent mutations (e.g., occurring during PCR amplification), or can bethe product of deliberate mutagenesis of a native sequence.

The invention thus provides isolated DNA sequences encoding polypeptidesof the invention, selected from: (a) DNA comprising the nucleotidesequence of SEQ ID NO:2; (b) DNA encoding the polypeptide of SEQ IDNO:3; (c) DNA capable of hybridization to a DNA of (a) or (b) underconditions of moderate stringency and which encode polypeptides of theinvention, wherein the polypeptides inhibit the growth of LNCaP cells ina colony-forming assay; (d) DNA capable of hybridization to a DNA of (a)or (b) under conditions of high stringency and which encodespolypeptides of the invention, wherein the polypeptides inhibit thegrowth of LNCaP cells in a colony-forming assay, and (e) DNA which isdegenerate as a result of the genetic code to a DNA defined in (a), (b),(c), or (d) and which encode polypeptides of the invention. Of course,polypeptides encoded by such DNA sequences are encompassed by theinvention.

As used herein, conditions of moderate stringency can be readilydetermined by those having ordinary skill in the art based on, forexample, the length of the DNA. The basic conditions are set forth by(Sambrook et al. Molecular Cloning: A Laboratory Manual, 3^(rd) ed.,Cold Spring Harbor Laboratory Press, (www.molecularcloning.com)), andinclude use of a prewashing solution for the nitrocellulose filters5×SSC, 0.5% SDS, 1.0 mM EDTA (pH 8.0), hybridization conditions of about50% formamide, 6×SSC at about 42° C. (or other similar hybridizationsolution, such as Stark's solution, in about 50% formamide at about 42°C.), and washing conditions of about 60° C., 0.5×SSC, 0.1% SDS.Conditions of high stringency can also be readily determined by theskilled artisan based on, for example, the length of the DNA. Generally,such conditions are defined as hybridization conditions as above, andwith washing at approximately 68° C., 0.2×SSC, 0.1% SDS. The skilledartisan will recognize that the temperature and wash solution saltconcentration can be adjusted as necessary according to factors such asthe length of the probe.

Also included as an embodiment of the invention is DNA encodingpolypeptide fragments and polypeptides comprising inactivatedN-glycosylation site(s), inactivated protease processing site(s), orconservative amino acid substitution(s), as described below.

In another embodiment, the nucleic acid molecules of the invention alsocomprise nucleotide sequences that are at least 80% identical to anative sequence (e.g., SEQ ID NO:2). Also contemplated are embodimentsin which a nucleic acid molecule comprises a sequence that is at least90% identical, at least 95% identical, at least 98% identical, at least99% identical, or at least 99.9% identical to a native sequence (e.g.,SEQ ID NO:2).

The percent identity may be determined by visual inspection andmathematical calculation. Alternatively, the percent identity of twonucleic acid sequences can be determined by comparing sequenceinformation using the GAP computer program, version 6.0 described by(Devereux et al., Nucl. Acids Res., 12:387 (1984)) and available fromthe University of Wisconsin Genetics Computer Group (UWGCG). Thepreferred default parameters for the GAP program include: (1) a unarycomparison matrix (containing a value of 1 for identities and 0 fornon-identities) for nucleotides, and the weighted comparison matrix of(Gribskov and Burgess, Nucl. Acids Res., 14:6745 (1986)), as describedby (Schwartz and Dayhoff, eds., Atlas of Protein Sequence and Structure,National Biomedical Research Foundation, pp. 353-358 (1979)); (2) apenalty of 3.0 for each gap and an additional 0.10 penalty for eachsymbol in each gap; and (3) no penalty for end gaps. Other programs usedby one skilled in the art of sequence comparison may also be used.

The invention also provides isolated nucleic acids useful in theproduction of polypeptides. Such polypeptides may be prepared by any ofa number of conventional techniques. A DNA sequence encoding a PMEPA1polypeptide, or desired fragment thereof may be subcloned into anexpression vector for production of the polypeptide or fragment. The DNAsequence advantageously is fused to a sequence encoding a suitableleader or signal peptide. Alternatively, the desired fragment may bechemically synthesized using known techniques. DNA fragments also may beproduced by restriction endonuclease digestion of a full length clonedDNA sequence, and isolated by electrophoresis on agarose gels. Ifnecessary, oligonucleotides that reconstruct the 5′ or 3′ terminus to adesired point may be ligated to a DNA fragment generated by restrictionenzyme digestion. Such oligonucleotides may additionally contain arestriction endonuclease cleavage site upstream of the desired codingsequence, and position an initiation codon (ATG) at the N-terminus ofthe coding sequence.

The well-known polymerase chain reaction (PCR) procedure also may beused to isolate and amplify a DNA sequence encoding a desired proteinfragment. Oligonucleotides that define the desired termini of the DNAfragment are employed as 5′ and 3′ primers. The oligonucleotides mayadditionally contain recognition sites for restriction endonucleases, tofacilitate insertion of the amplified DNA fragment into an expressionvector. PCR techniques are described in (Saiki et al., Science, 239:487(1988)); (Wu et al., Recombinant DNA Methodology, eds., Academic Press,Inc., San Diego, pp. 189-196 (1989)); and (Innis et al., PCR Protocols:A Guide to Methods and Applications, eds., Academic Press, Inc. (1990)).

Polypeptides and Fragments Thereof

The invention encompasses polypeptides and fragments thereof in variousforms, including those that are naturally occurring or produced throughvarious techniques such as procedures involving recombinant DNAtechnology. Such forms include, but are not limited to, derivatives,variants, and oligomers, as well as fusion proteins or fragmentsthereof.

Polypeptides and Fragments Thereof

The polypeptides of the invention include full length proteins encodedby the nucleic acid sequences set forth above. Particularly preferredpolypeptides comprise the amino acid sequence of SEQ ID NO:3.

As discussed in Example 8, SEQ ID NO:3 shares 83% identity to a NEDD4 WWbinding protein and contains two PY motifs, i.e., PPPY (SEQ ID NO:80)(“PY1”) and PPTY (SEQ ID NO:81) (“PY2”). The PPXY motif, where X can beany amino acid, has been shown to facilitate binding with WWdomain-containing proteins. We demonstrate in the Examples that PMEPA1binds to the NEDD4 protein, which contains WW domains. NEDD4 is aubiquitin-protein ligase (E3) that is involved in theubiquitin-dependent proteasome-mediated protein degradation pathway.

Assays for determining whether a polypeptide, such as PMEPA1, binds toother proteins having a WW domain are well-known in the art and includestrategies such as combinatorial peptide libraries, affinitychromatography, expression library screening, and yeast two-hybridscreening (Kay et al. (2000) FEBS Lett., 480:55-62; Frederick et al.(1999) Mol. Cell. Biol., 19: 2330-2337; Dai and Pendergast (1995) GenesDev., 9:2569-2582; Kitamura et al. (1996) Biochem. Biophys. Res.Commun., 219:509-514; Richard et al. (1995) Mol. Cell. Biol. 15:186-197;and Sudol (1994) Oncogene 9:2145-2152).

The experimental data presented in the Examples show that PMEPA1negatively regulates cancer cell growth. Loss of such function favorstumorigenesis or progression of existing disease. Thus, PMEPA1 maysuppress tumorigenesis or cancer progression by interacting with WWdomain-containing molecules. The homology of PMEPA1 to the NEDD4-bindingprotein and the ability of PMEPA1 to bind NEDD4 indicates that PMEPA1may regulate protein turnover via ubiquitinylation and proteasomepathways in the cell. This mechanism is, of course, merely proposed.Moreover, it is not the only mechanism by which PMEPA1 may exert itsfunction. The present invention is not limited to any particularmechanism of PMEPA1 activity.

In one embodiment, a polypeptide of the invention comprises an aminoacid sequence as set out in SEQ ID NO:3. In another embodiment, thepolypeptide comprises an amino acid sequence substantially as set out inSEQ ID NO:3. In yet another embodiment, the polypeptide comprises anamino acid sequence that is at least 80%, 90%, 95%, 96%, 97%, 98%, 99%,OR 99.9% identical to SEQ ID NO:3, and preferably the polypeptideinhibits prostate cancer cell growth, as demonstrated, for example, in acolony-forming assay, such as the one described in Example 12.Inhibiting cell growth refers to a decrease in cell growth in thepresence of a PMEPA1 polypeptide, relative to the cell growth in theabsence of the PMEPA1 polypeptide. Alternatively, if a cell has a basallevel of PMEPA1 polypeptide expression, it refers to a decrease in cellgrowth in the presence of increased levels of PMEPA1 polypeptide,relative to cell growth in the presence of the basal level of PMEPA1polypeptide. Cell growth can be measured using conventional assays, suchas the colony-forming assay described in the examples. As discussed infurther detail below, these polypeptides may be produced by recombinantDNA techniques. Percent identity may be determined by visual inspectionand mathematical calculation. Alternatively, the percent identity of twoprotein sequences can be determined by comparing sequence informationusing the GAP computer program, based on the algorithm of (Needleman andWunsch, J. Mol. Bio., 48:443 (1970)) and available from the Universityof Wisconsin Genetics Computer Group (UWGCG). The preferred defaultparameters for the GAP program include: (1) a scoring matrix, blosum62,as described by (Henikoff and Henikoff Proc. Natl. Acad. Sci. USA,89:10915 (1992)); (2) a gap weight of 12; (3) a gap length weight of 4;and (4) no penalty for end gaps. Other programs used by one skilled inthe art of sequence comparison may also be used.

The polypeptides of the invention may be membrane bound or they may besecreted and thus soluble. Soluble polypeptides are capable of beingsecreted from the cells in which they are expressed. In general, solublepolypeptides may be identified (and distinguished from non-solublemembrane-bound counterparts) by separating intact cells which expressthe desired polypeptide from the culture medium, e.g., bycentrifugation, and assaying the medium (supernatant) for the presenceof the desired polypeptide. The presence of polypeptide in the mediumindicates that the polypeptide was secreted from the cells and thus is asoluble form of the protein.

In one embodiment, the soluble polypeptides and fragments thereofcomprise all or part of the extracellular domain, but lack thetransmembrane region that would cause retention of the polypeptide on acell membrane. A soluble polypeptide may include the cytoplasmic domain,or a portion thereof, as long as the polypeptide is secreted from thecell in which it is produced.

In general, the use of soluble forms is advantageous for certainapplications. Purification of the polypeptides from recombinant hostcells is facilitated, since the soluble polypeptides are secreted fromthe cells. Further, soluble polypeptides are generally more suitable forintravenous administration.

The invention also provides polypeptides and fragments of theextracellular domain that retain a desired biological activity. Such afragment may be a soluble polypeptide, as described above.

Also provided herein are polypeptide fragments comprising at least 20,or at least 30, contiguous amino acids of the sequence of SEQ ID NO:3.Fragments derived from the cytoplasmic domain find use in studies ofsignal transduction, and in regulating cellular processes associatedwith transduction of biological signals. Polypeptide fragments also maybe employed as immunogens, in generating antibodies.

Variants

Naturally occurring variants as well as derived variants of thepolypeptides and fragments are provided herein.

The variants of the invention include, for example, those that resultfrom alternate mRNA splicing events or from proteolytic cleavage.Alternate splicing of mRNA may, for example, yield a truncated butbiologically active protein, such as a naturally occurring soluble formof the protein. Variations attributable to proteolysis include, forexample, differences in the N- or C-termini upon expression in differenttypes of host cells, due to proteolytic removal of one or more terminalamino acids from the protein (generally from 1-5 terminal amino acids).Proteins in which differences in amino acid sequence are attributable togenetic polymorphism (allelic variation among individuals producing theprotein) are also contemplated herein.

Additional variants within the scope of the invention includepolypeptides that may be modified to create derivatives thereof byforming covalent or aggregative conjugates with other chemical moieties,such as glycosyl groups, lipids, phosphate, acetyl groups and the like.Covalent derivatives may be prepared by linking the chemical moieties tofunctional groups on amino acid side chains or at the N-terminus orC-terminus of a polypeptide. Conjugates comprising diagnostic(detectable) or therapeutic agents attached thereto are contemplatedherein, as discussed in more detail below.

Other derivatives include covalent or aggregative conjugates of thepolypeptides with other proteins or polypeptides, such as by synthesisin recombinant culture as N-terminal or C-terminal fusions. Examples offusion proteins are discussed below in connection with oligomers.Further, fusion proteins can comprise peptides added to facilitatepurification and identification. Such peptides include, for example,poly-His or the antigenic identification peptides described in U.S. Pat.No. 5,011,912 and in (Hopp et al., Bio/Technology, 6:1204 (1988)). Onesuch peptide is the FLAG® peptide, Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys, (SEQID NO:4) which is highly antigenic and provides an epitope reversiblybound by a specific monoclonal antibody, enabling rapid assay and facilepurification of expressed recombinant protein. A murine hybridomadesignated 4E11 produces a monoclonal antibody that binds the FLAG®peptide in the presence of certain divalent metal cations, as describedin U.S. Pat. No. 5,011,912, hereby incorporated by reference. The 4E11hybridoma cell line has been deposited with the American Type CultureCollection under accession no. HB 9259. Monoclonal antibodies that bindthe FLAG® peptide are available from Eastman Kodak Co., ScientificImaging Systems Division, New Haven, Conn.

Among the variant polypeptides provided herein are variants of nativepolypeptides that retain one or more activities associated with afull-length, wild-type, PMEPA1 protein. As one example, such variants oranalogs that have the desired immunogenicity or antigenicity can beused, for example, in immunoassays, for immunization, for inhibition ofPMEPA1 activity, etc. Variants or analogs that retain, or alternativelylack or inhibit, a desired PMEPA1 property of interest can be used asinducers, or inhibitors, respectively, of such property and itsphysiological correlates. These PMEPA1 properties include, but are notlimited to, binding to a WW domain-containing protein or other PMEPA1binding partner, inhibiting cancer cell proliferation, inhibiting theexpression of an androgen receptor, and modulating the expression of agene whose transcription is regulated by the androgen receptor. Bindingaffinity can be measured by conventional procedures, e.g., as describedin U.S. Pat. No. 5,512,457 and as set forth below. Variants or analogsof PMEPA1 can be tested for the desired activity by procedures known inthe art, including but not limited to, the assays described in theExamples.

In one embodiment, the PMEPA1 variants contain at least one mutationand/or deletion in the at least one of the PY motifs of PMEPA1. Thesevariants can be used, for example, in the treatment of hypoproliferativedisorders. In addition, these variants can be used as immunogens togenerate antibodies.

Variants include polypeptides that are substantially homologous to thenative form, but which have an amino acid sequence different from thatof the native form because of one or more deletions, insertions orsubstitutions. Particular embodiments include, but are not limited to,polypeptides that comprise from one to ten deletions, insertions orsubstitutions of amino acid residues, when compared to a nativesequence.

A given amino acid may be replaced, for example, by a residue havingsimilar physiochemical characteristics. Examples of such conservativesubstitutions include substitution of one aliphatic residue for another,such as Ile, Val, Leu, or Ala for one another; substitutions of onepolar residue for another, such as between Lys and Arg, Glu and Asp, orGln and Asn; or substitutions of one aromatic residue for another, suchas Phe, Trp, or Tyr for one another. Other conservative substitutions,e.g., involving substitutions of entire regions having similarhydrophobicity characteristics, are well known.

Similarly, the DNAs of the invention include variants that differ from anative DNA sequence because of one or more deletions, insertions orsubstitutions, but that encode a biologically active polypeptide.

The invention further includes polypeptides of the invention with orwithout associated native-pattern glycosylation. Polypeptides expressedin yeast or mammalian expression systems (e.g., COS-1 or COS-7 cells)can be similar to or significantly different from a native polypeptidein molecular weight and glycosylation pattern, depending upon the choiceof expression system. Expression of polypeptides of the invention inbacterial expression systems, such as E. coli, provides non-glycosylatedmolecules. Further, a given preparation may include multipledifferentially glycosylated species of the protein. Glycosyl groups canbe removed through conventional methods, in particular those utilizingglycopeptidase. In general, glycosylated polypeptides of the inventioncan be incubated with a molar excess of glycopeptidase (BoehringerMannheim).

Correspondingly, similar DNA constructs that encode various additions orsubstitutions of amino acid residues or sequences, or deletions ofterminal or internal residues or sequences are encompassed by theinvention. For example, N-glycosylation sites in the polypeptideextracellular domain can be modified to preclude glycosylation, allowingexpression of a reduced carbohydrate analog in mammalian and yeastexpression systems. N-glycosylation sites in eukaryotic polypeptides arecharacterized by an amino acid triplet Asn-X-Y, wherein X is any aminoacid and Y is Ser or Tbr. Appropriate substitutions, additions, ordeletions to the nucleotide sequence encoding these triplets will resultin prevention of attachment of carbohydrate residues at the Asn sidechain. Alteration of a single nucleotide, chosen so that Asn is replacedby a different amino acid, for example, is sufficient to inactivate anN-glycosylation site. Alternatively, the Ser or Thr can by replaced withanother amino acid, such as Ala. Known procedures for inactivatingN-glycosylation sites in proteins include those described in U.S. Pat.No. 5,071,972 and EP 276,846, hereby incorporated by reference.

In another example of variants, sequences encoding Cys residues that arenot essential for biological activity can be altered to cause the Cysresidues to be deleted or replaced with other amino acids, preventingformation of incorrect intramolecular disulfide bridges upon folding orrenaturation.

Other variants are prepared by modification of adjacent dibasic aminoacid residues, to enhance expression in yeast systems in which KEX2protease activity is present. EP 212,914 discloses the use ofsite-specific mutagenesis to inactivate KEX2 protease processing sitesin a protein. KEX2 protease processing sites are inactivated bydeleting, adding or substituting residues to alter Arg-Arg, Arg-Lys, andLys-Arg pairs to eliminate the occurrence of these adjacent basicresidues. Lys-Lys pairings are considerably less susceptible to KEX2cleavage, and conversion of Arg-Lys or Lys-Arg to Lys-Lys represents aconservative and preferred approach to inactivating KEX2 sites.

Production of Polypeptides and Fragments Thereof

Expression, isolation and purification of the polypeptides and fragmentsof the invention may be accomplished by any suitable technique,including but not limited to the following:

Expression Systems

The present invention also provides recombinant cloning and expressionvectors containing DNA, as well as host cell containing the recombinantvectors. Expression vectors comprising DNA may be used to prepare thepolypeptides or fragments of the invention encoded by the DNA. A methodfor producing polypeptides comprises culturing host cells transformedwith a recombinant expression vector encoding the polypeptide, underconditions that promote expression of the polypeptide, then recoveringthe expressed polypeptides from the culture. The skilled artisan willrecognize that the procedure for purifying the expressed polypeptideswill vary according to such factors as the type of host cells employed,and whether the polypeptide is membrane-bound or a soluble form that issecreted from the host cell.

Any suitable expression system may be employed. The vectors include aDNA encoding a polypeptide or fragment of the invention, operably linkedto suitable transcriptional or translational regulatory nucleotidesequences, such as those derived from a mammalian, microbial, viral, orinsect gene. Examples of regulatory sequences include transcriptionalpromoters, operators, or enhancers, an mRNA ribosomal binding site, andappropriate sequences which control transcription and translationinitiation and termination. Nucleotide sequences are operably linkedwhen the regulatory sequence functionally relates to the DNA sequence.Thus, a promoter nucleotide sequence is operably linked to a DNAsequence if the promoter nucleotide sequence controls the transcriptionof the DNA sequence. An origin of replication that confers the abilityto replicate in the desired host cells, and a selection gene by whichtransformants are identified, are generally incorporated into theexpression vector.

In addition, a sequence encoding an appropriate signal peptide (nativeor heterologous) can be incorporated into expression vectors. A DNAsequence for a signal peptide (secretory leader) may be fused in frameto the nucleic acid sequence of the invention so that the DNA isinitially transcribed, and the mRNA translated, into a fusion proteincomprising the signal peptide. A signal peptide that is functional inthe intended host cells promotes extracellular secretion of thepolypeptide. The signal peptide is cleaved from the polypeptide uponsecretion of polypeptide from the cell.

Suitable host cells for expression of polypeptides include prokaryotes,yeast or higher eukaryotic cells. Mammalian or insect cells aregenerally preferred for use as host cells. Appropriate cloning andexpression vectors for use with bacterial, fungal, yeast, and mammaliancellular hosts are described, for example, in (Pouwels et al. CloningVectors: A Laboratory Manual, Elsevier, New York, (1985)). Cell-freetranslation systems could also be employed to produce polypeptides usingRNAs derived from DNA constructs disclosed herein.

Prokaryotic Systems

Prokaryotes include gram-negative or gram-positive organisms. Suitableprokaryotic host cells for transformation include, for example, E. coli,Bacillus subtilis, Salmonella typhimurium, and various other specieswithin the genera Pseudomonas, Streptomyces, and Staphylococcus. In aprokaryotic host cell, such as E. coli, a polypeptide may include anN-terminal methionine residue to facilitate expression of therecombinant polypeptide in the prokaryotic host cell. The N-terminal Metmay be cleaved from the expressed recombinant polypeptide.

Expression vectors for use in prokaryotic host cells generally compriseone or more phenotypic selectable marker genes. A phenotypic selectablemarker gene is, for example, a gene encoding a protein that confersantibiotic resistance or that supplies an autotrophic requirement.Examples of useful expression vectors for prokaryotic host cells includethose derived from commercially available plasmids such as the cloningvector pBR322 (ATCC 37017). pBR322 contains genes for ampicillin andtetracycline resistance and thus provides simple means for identifyingtransformed cells. An appropriate promoter and a DNA sequence areinserted into the pBR322 vector. Other commercially available vectorsinclude, for example, pKK223-3 (Pharmacia Fine Chemicals, Uppsala,Sweden) and pGEM1 (Promega Biotec, Madison, Wis., USA).

Promoter sequences commonly used for recombinant prokaryotic host cellexpression vectors include β-lactamase (penicillinase), lactose promotersystem (Chang et al., Nature 275:615 (1978); and (Goeddel et al., Nature281:544 (1979)), tryptophan (trp) promoter system (Goeddel et al., Nucl.Acids Res. 8:4057 (1980); and EP-A-36776) and tac promoter (Maniatis,Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory,p. 412 (1982)). A particularly useful prokaryotic host cell expressionsystem employs a phage λP_(L) promoter and a c1857ts thermolabilerepressor sequence. Plasmid vectors available from the American TypeCulture Collection which incorporate derivatives of the λP_(L) promoterinclude plasmid pHUB2 (resident in E. coli strain JMB9, ATCC 37092) andpPLc28 (resident in E. coli RR1, ATCC 53082).

Yeast Systems

Alternatively, the polypeptides may be expressed in yeast host cells,preferably from the Saccharomyces genus (e.g., S. cerevisiae). Othergenera of yeast, such as Pichia or Kluyveromyces, may also be employed.Yeast vectors will often contain an origin of replication sequence froma 2μ yeast plasmid, an autonomously replicating sequence (ARS), apromoter region, sequences for polyadenylation, sequences fortranscription termination, and a selectable marker gene. Suitablepromoter sequences for yeast vectors include, among others, promotersfor metallothionein, 3-phosphoglycerate kinase (Hitzeman et al., J.Biol. Chem. 255:2073 (1980)) or other glycolytic enzymes (Hess et al., JAdv. Enzyme Reg. 7:149 (1968)); and (Holland et al., Biochem. 17:4900(1978)), such as enolase, glyceraldehyde-3-phosphate dehydrogenase,hexokinase, pyruvate decarboxylase, phosphofructokinase,glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvatekinase, triosephosphate isomerase, phospho-glucose isomerase, andglucokinase. Other suitable vectors and promoters for use in yeastexpression are further described in (Hitzeman, EPA-73,657). Anotheralternative is the glucose-repressible ADH2 promoter described by(Russell et al., J. Biol. Chem. 258:2674 (1982)) and (Beier et al.,Nature 300:724 (1982)). Shuttle vectors replicable in both yeast and E.coli may be constructed by inserting DNA sequences from pBR322 forselection and replication in E. coli (Ampr gene and origin ofreplication) into the above-described yeast vectors.

The yeast α-factor leader sequence may be employed to direct secretionof the polypeptide. The α-factor leader sequence is often insertedbetween the promoter sequence and the structural gene sequence. See,e.g., (Kurjan et al., Cell 30:933 (1982)) and (Bitter et al., Proc.Natl. Acad. Sci. USA 81:5330 (1984)). Other leader sequences suitablefor facilitating secretion of recombinant polypeptides from yeast hostsare known to those of skill in the art. A leader sequence may bemodified near its 3′ end to contain one or more restriction sites. Thiswill facilitate fusion of the leader sequence to the structural gene.

Yeast transformation protocols are known to those of skill in the art.One such protocol is described by (Hinnen et al., Proc. Natl. Acad. Sci.USA 75:1929 (1978)). The Hinnen et al. protocol selects for Trp⁺transformants in a selective medium, wherein the selective mediumconsists of 0.67% yeast nitrogen base, 0.5% casamino acids, 2% glucose,10 mg/ml adenine and 20 mg/ml uracil.

Yeast host cells transformed by vectors containing an ADH2 promotersequence may be grown for inducing expression in a “rich” medium. Anexample of a rich medium is one consisting of 1% yeast extract, 2%peptone, and 1% glucose supplemented with 80 mg/ml adenine and 80 mg/mluracil. Derepression of the ADH2 promoter occurs when glucose isexhausted from the medium.

Mammalian or Insect Systems

Mammalian or insect host cell culture systems also may be employed toexpress recombinant polypeptides. Bacculovirus systems for production ofheterologous proteins in insect cells are reviewed by (Luckow andSummers, Bio/Technology, 6:47 (1988)). Established cell lines ofmammalian origin also may be employed. Examples of suitable mammalianhost cell lines include the COS-7 line of monkey kidney cells (ATCC CRL1651) (Gluzman et al., Cell 23:175 (1981)), L cells, C127 cells, 3T3cells (ATCC CCL 163), Chinese hamster ovary (CHO) cells, HeLa cells, andBHK (ATCC CRL 10) cell lines, and the CV1/EBNA cell line derived fromthe African green monkey kidney cell line CV1 (ATCC CCL 70) as describedby (McMahan et al., EMBO J, 10: 2821 (1991)).

Established methods for introducing DNA into mammalian cells have beendescribed (Kaufman, R. J., Large Scale Mammalian Cell Culture, pp. 15-69(1990)). Additional protocols using commercially available reagents,such as Lipofectamine lipid reagent (Gibco/BRL) or Lipofectamine-Pluslipid reagent, can be used to transfect cells (Felgner et al., Proc.Natl. Acad. Sci. USA 84:7413-7417 (1987)). In addition, electroporationcan be used to transfect mammalian cells using conventional procedures,such as those in (Sambrook et al., Molecular Cloning: A LahoratoryManual, 2 ed. Vol. 1-3, Cold Spring Harbor Laboratory Press (1989)).Selection of stable transformants can be performed using methods knownin the art, such as, for example, resistance to cytotoxic drugs.(Kaufman et al., Meth. in Enzymology 185:487-511 (1990)), describesseveral selection schemes, such as dihydrofolate reductase (DHFR)resistance. A suitable host strain for DHFR selection can be CHO strainDX-B 11, which is deficient in DHFR (Urlaub and Chasin, Proc. Natl.Acad. Sci. USA 77:4216-4220 (1980)). A plasmid expressing the DHFR cDNAcan be introduced into strain DX-B 11, and only cells that contain theplasmid can grow in the appropriate selective media. Other examples ofselectable markers that can be incorporated into an expression vectorinclude cDNAs conferring resistance to antibiotics, such as G418 andhygromycin B. Cells harboring the vector can be selected on the basis ofresistance to these compounds.

Transcriptional and translational control sequences for mammalian hostcell expression vectors can be excised from viral genomes. Commonly usedpromoter sequences and enhancer sequences are derived from polyomavirus, adenovirus 2, simian virus 40 (SV40), and human cytomegalovirus.DNA sequences derived from the SV40 viral genome, for example, SV40origin, early and late promoter, enhancer, splice, and polyadenylationsites can be used to provide other genetic elements for expression of astructural gene sequence in a mammalian host cell. Viral early and latepromoters are particularly useful because both are easily obtained froma viral genome as a fragment, which can also contain a viral origin ofreplication (Fiers et al., Nature 273:113 (1978)); (Kaufman, Meth. inEnzymology (1990)). Smaller or larger SV40 fragments can also be used,provided the approximately 250 bp sequence extending from the Hind IIIsite toward the Bgl I site located in the SV40 viral origin ofreplication site is included.

Additional control sequences shown to improve expression of heterologousgenes from mammalian expression vectors include such elements as theexpression augmenting sequence element (EASE) derived from CHO cells(Morris et al., Animal Cell Technology, pp. 529-534 and PCT ApplicationWO 97/25420 (1997)) and the tripartite leader (TPL) and VA gene RNAsfrom Adenovirus 2 (Gingeras et al., J. Biol. Chem. 257:13475-13491(1982)). The internal ribosome entry site (IRES) sequences of viralorigin allows dicistronic mRNAs to be translated efficiently (Oh andSarnow, Current Opinion in Genetics and Development 3:295-300 (1993));(Ramesh et al., Nucleic Acids Research 24:2697-2700 (1996)). Expressionof a heterologous cDNA as part of a dicistronic mRNA followed by thegene for a selectable marker (e.g. DHFR) has been shown to improvetransfectability of the host and expression of the heterologous cDNA(Kaufman, Meth. in Enzymology (1990)). Exemplary expression vectors thatemploy dicistronic mRNAs are pTR-DC/GFP described by (Mosser et al.,Biotechniques 22:150-161 (1997)), and p2A5I described by (Morris et al.,Animal Cell Technology, pp. 529-534 (1997)).

A useful high expression vector, pCAVNOT, has been described by (Mosleyet al., Cell 59:335-348 (1989)). Other expression vectors for use inmammalian host cells can be constructed as disclosed by (Okayama andBerg, Mol. Cell. Biol. 3:280 (1983)). A useful system for stable highlevel expression of mammalian cDNAs in C127 murine mammary epithelialcells can be constructed substantially as described by (Cosman et al.,Mol. Immunol. 23:935 (1986)). A useful high expression vector, PMLSVN1/N4, described by (Cosman et al., Nature 312:768 (1984)), has beendeposited as ATCC 39890. Additional useful mammalian expression vectorsare described in EP-A-0367566, and in WO 91/18982, incorporated byreference herein. In yet another alternative, the vectors can be derivedfrom retroviruses.

Another useful expression vector, pFLAG®, can be used. FLAG® technologyis centered on the fusion of a low molecular weight (1 kD), hydrophilic,FLAG® marker peptide to the N-terminus of a recombinant proteinexpressed by pFLAG® expression vectors. pDC311 is another specializedvector used for expressing proteins in CHO cells. pDC311 ischaracterized by a bicistronic sequence containing the gene of interestand a dihydrofolate reductase (DHFR) gene with an internal ribosomebinding site for DHFR translation, an expression augmenting sequenceelement (EASE), the human CMV promoter, a tripartite leader sequence,and a polyadenylation site.

Purification

The invention also includes methods of isolating and purifying thepolypeptides and fragments thereof.

Isolation and Purification

The “isolated” polypeptides or fragments thereof encompassed by thisinvention are polypeptides or fragments that are not in an environmentidentical to an environment in which it or they can be found in nature.The “purified” polypeptides or fragments thereof encompassed by thisinvention are essentially free of association with other proteins orpolypeptides, for example, as a purification product of recombinantexpression systems such as those described above or as a purifiedproduct from a non-recombinant source such as naturally occurring cellsand/or tissues.

In one preferred embodiment, the purification of recombinantpolypeptides or fragments can be accomplished using fusions ofpolypeptides or fragments of the invention to another polypeptide to aidin the purification of polypeptides or fragments of the invention.

With respect to any type of host cell, as is known to the skilledartisan, procedures for purifying a recombinant polypeptide or fragmentwill vary according to such factors as the type of host cells employedand whether or not the recombinant polypeptide or fragment is secretedinto the culture medium.

In general, the recombinant polypeptide or fragment can be isolated fromthe host cells if not secreted, or from the medium or supernatant ifsoluble and secreted, followed by one or more concentration,salting-out, ion exchange, hydrophobic interaction, affinitypurification or size exclusion chromatography steps. As to specific waysto accomplish these steps, the culture medium first can be concentratedusing a commercially available protein concentration filter, forexample, an Amicon or Millipore Pellicon ultrafiltration unit. Followingthe concentration step, the concentrate can be applied to a purificationmatrix such as a gel filtration medium. Alternatively, an anion exchangeresin can be employed, for example, a matrix or substrate having pendantdiethylaminoethyl (DEAE) groups. The matrices can be acrylamide,agarose, dextran, cellulose or other types commonly employed in proteinpurification. Alternatively, a cation exchange step can be employed.Suitable cation exchangers include various insoluble matrices comprisingsulfopropyl or carboxymethyl groups. In addition, a chromatofocusingstep can be employed. Alternatively, a hydrophobic interactionchromatography step can be employed. Suitable matrices can be phenyl oroctyl moieties bound to resins. In addition, affinity chromatographywith a matrix which selectively binds the recombinant protein can beemployed. Examples of such resins employed are lectin columns, dyecolumns, and metal-chelating columns. Finally, one or morereversed-phase high performance liquid chromatography (RP-HPLC) stepsemploying hydrophobic RP-HPLC media, (e.g., silica gel or polymer resinhaving pendant methyl, octyl, octyldecyl or other aliphatic groups) canbe employed to further purify the polypeptides. Some or all of theforegoing purification steps, in various combinations, are well knownand can be employed to provide an isolated and purified recombinantprotein.

It is also possible to utilize an affinity column comprising apolypeptide-binding protein of the invention, such as a monoclonalantibody generated against polypeptides of the invention, toaffinity-purify expressed polypeptides. These polypeptides can beremoved from an affinity column using conventional techniques, e.g., ina high salt elution buffer and then dialyzed into a lower salt bufferfor use or by changing pH or other components depending on the affinitymatrix utilized, or be competitively removed using the naturallyoccurring substrate of the affinity moiety, such as a polypeptidederived from the invention.

In this aspect of the invention, polypeptide-binding proteins, such asthe anti-polypeptide antibodies of the invention or other proteins thatmay interact with the polypeptide of the invention, can be bound to asolid phase support such as a column chromatography matrix or a similarsubstrate suitable for identifying, separating, or purifying cells thatexpress polypeptides of the invention on their surface. Adherence ofpolypeptide-binding proteins of the invention to a solid phasecontacting surface can be accomplished by any means, for example,magnetic microspheres can be coated with these polypeptide-bindingproteins and held in the incubation vessel through a magnetic field.Suspensions of cell mixtures are contacted with the solid phase that hassuch polypeptide-binding proteins thereon. Cells having polypeptides ofthe invention on their surface bind to the fixed polypeptide-bindingprotein and unbound cells then are washed away. This affinity-bindingmethod is useful for purifying, screening, or separating suchpolypeptide-expressing cells from solution. Methods of releasingpositively selected cells from the solid phase are known in the art andencompass, for example, the use of enzymes. Such enzymes are preferablynon-toxic and non-injurious to the cells and are preferably directed tocleaving the cell-surface binding partner.

Alternatively, mixtures of cells suspected of containingpolypeptide-expressing cells of the invention first can be incubatedwith a biotinylated polypeptide-binding protein of the invention.Incubation periods are typically at least one hour in duration to ensuresufficient binding to polypeptides of the invention. The resultingmixture then is passed through a column packed with avidin-coated beads,whereby the high affinity of biotin for avidin provides the binding ofthe polypeptide-binding cells to the beads. Use of avidin-coated beadsis known in the art. See (Berenson, et al. J. Cell. Biochem., 10D:239(1986)). Wash of unbound material and the release of the bound cells isperformed using conventional methods.

The desired degree of purity depends on the intended use of the protein.A relatively high degree of purity is desired when the polypeptide is tobe administered in vivo, for example. In such a case, the polypeptidesare purified such that no protein bands corresponding to other proteinsare detectable upon analysis by SDS-polyacrylamide gel electrophoresis(SDS-PAGE). It will be recognized by one skilled in the pertinent fieldthat multiple bands corresponding to the polypeptide may be visualizedby SDS-PAGE, due to differential glycosylation, differentialpost-translational processing, and the like. Most preferably, thepolypeptide of the invention is purified to substantial homogeneity, asindicated by a single protein band upon analysis by SDS-PAGE. Theprotein band may be visualized by silver staining, Coomassie bluestaining, or (if the protein is radiolabeled) by autoradiography.

Production of Antibodies

Antibodies that are immunoreactive with the polypeptides of theinvention are provided herein. Such antibodies specifically bind to thepolypeptides via the antigen-binding sites of the antibody (as opposedto non-specific binding). Thus, the polypeptides, fragments, variants,fusion proteins, etc., as set forth above may be employed as“immunogens” in producing antibodies immunoreactive therewith. Morespecifically, the polypeptides, fragment, variants, fusion proteins,etc. contain antigenic determinants or epitopes that elicit theformation of antibodies.

These antigenic determinants or epitopes can be either linear orconformational (discontinuous). Linear epitopes are composed of a singlesection of amino acids of the polypeptide, while conformational ordiscontinuous epitopes are composed of amino acids sections fromdifferent regions of the polypeptide chain that are brought into closeproximity upon protein folding (C. A. Janeway, Jr. and P. Travers,Immuno Biology 3:9, Garland Publishing Inc., 2nd ed. (1996)). Becausefolded proteins have complex surfaces, the number of epitopes availableis quite numerous; however, due to the conformation of the protein andsteric hinderances, the number of antibodies that actually bind to theepitopes is less than the number of available epitopes (C. A. Janeway,Jr. and P. Travers, Immuno Biology 2:14, Garland Publishing Inc., 2nded. (1996)). Epitopes may be identified by any of the methods known inthe art.

Thus, one aspect of the present invention relates to the antigenicepitopes of the polypeptides of the invention. Such epitopes are usefulfor raising antibodies, in particular monoclonal antibodies, asdescribed in more detail below. Additionally, epitopes from thepolypeptides of the invention can be used as research reagents, inassays, and to purify specific binding antibodies from substances suchas polyclonal sera or supernatants from cultured hybridomas. Suchepitopes or variants thereof can be produced using techniques well knownin the art such as solid-phase synthesis, chemical or enzymatic cleavageof a polypeptide, or using recombinant DNA technology.

As to the antibodies that can be elicited by the epitopes of thepolypeptides of the invention, whether the epitopes have been isolatedor remain part of the polypeptides, both polyclonal and monoclonalantibodies may be prepared by conventional techniques. See, for example,(Kennet et al., Monoclonal Antibodies, Hybridomas: A New Dimension inBiological Analyses, eds., Plenum Press, New York (1980); and Harlow andLand, Antibodies: A Laboratory Manual, eds., Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., (1988)).

Hybridoma cell lines that produce monoclonal antibodies specific for thepolypeptides of the invention are also contemplated herein. Suchhybridomas may be produced and identified by conventional techniques.One method for producing such a hybridoma cell line comprises immunizingan animal with a polypeptide; harvesting spleen cells from the immunizedanimal; fusing said spleen cells to a myeloma cell line, therebygenerating hybridoma cells; and identifying a hybridoma cell line thatproduces a monoclonal antibody that binds the polypeptide. Themonoclonal antibodies may be recovered by conventional techniques.

The monoclonal antibodies of the present invention include chimericantibodies, e.g., humanized versions of murine monoclonal antibodies.Such humanized antibodies may be prepared by known techniques and offerthe advantage of reduced immunogenicity when the antibodies areadministered to humans. In one embodiment, a humanized monoclonalantibody comprises the variable region of a murine antibody (or just theantigen binding site thereof) and a constant region derived from a humanantibody. Alternatively, a humanized antibody fragment may comprise theantigen binding site of a murine monoclonal antibody and a variableregion fragment (lacking the antigen-binding site) derived from a humanantibody. Procedures for the production of chimeric and furtherengineered monoclonal antibodies include those described in (Riechmannet al., Nature 332:323 (1988), Liu et al., PNAS 84:3439 (1987), Larricket al., Bio/Technology 7:934 (1989), and Winter and Harris, TIPS 14:139(May 1993)). Procedures to generate antibodies transgenically can befound in GB 2,272,440, U.S. Pat. Nos. 5,569,825 and 5,545,806 andrelated patents claiming priority therefrom, all of which areincorporated by reference herein.

Antigen-binding fragments of the antibodies, which may be produced byconventional techniques, are also encompassed by the present invention.Examples of such fragments include, but are not limited to, Fab andF(ab′)₂ fragments. Antibody fragments and derivatives produced bygenetic engineering techniques are also provided.

In one embodiment, the antibodies are specific for the polypeptides ofthe present invention and do not cross-react with other proteins.Screening procedures by which such antibodies may be identified are wellknown, and may involve immunoaffinity chromatography, for example.

The following examples further illustrate preferred aspects of theinvention.

EXAMPLE 1

Cell Culture and Androgen Stimulation

LNCaP cells (American Type Culture Collection, Rockville, Md.) were usedfor SAGE analysis of ARGs. LNCaP cells were maintained in RPMI 1640(Life Technologies, Inc., Gaithersburg, Md.) supplemented with 10% fetalbovine serum (FBS, Life Technologies, Inc., Gaithersburg, Md.) andexperiments were performed on cells between passages 20 and 30. For thestudies of androgen regulation, charcoal/dextran stripped androgen-freeFBS (cFBS, Gemini Bio-Products, Inc., Calabasas, Calif.) was used. LNCaPcells were cultured first in RPMI 1640 with 10% cFBS for 5 days and thenstimulated with 10-8 M of non-metabolizable androgen analog, R1881(DUPONT, Boston, Mass.) for 24 hours. LNCaP cells identically treatedbut without R1881 treatment served as control. Cells were harvested atindicated time and polyA+ RNA was double-selected with Fast Track kit(Invitrogene). The quality of polyA+ was checked by Northernhybridization analysis.

EXAMPLE 2

SAGE Analysis

Two SAGE libraries (library LNCaP-C and library LNCaP-T) were generatedaccording to the procedure described previously Velculescu et al. (30).Briefly, biotinylated oligo dT primed cDNA was prepared from fivemicrograms of polyA+ RNA from R1881 treated and control LNCaP cells andbiotinylated cDNA was captured on strepravidin coated magnetic beads(Dynal Corporation, MI). cDNA bound to the magnetic beads were digestedby NlaIII followed by ligation to synthetic linkers containing a sitefor anchoring enzyme, NlaIII and a site for tagging enzyme BsmF1. Therestriction digestion of ligated products with BsmF1 resulted in thecapture of 10-11 bp sequences termed as “tags” representing signaturesequence of unique cDNAs. A multi-step strategy combining ligation, PCR,enzymatic digestion and gel purification yielded two tags linkedtogether termed as “ditags.” Ditags were concatamerized, purified andcloned in plasmid pZero cloning vector (Invitrogen, Calif.). The clonescontaining concatamers were screened by PCR and sequenced. The sequenceand the occurrence of each of the SAGE tags was determined using theSAGE software kindly provided by Dr. Kenneth W. Kinzler (Johns HopkinsUniversity School of Medicine, Baltimore, Md.). All the SAGE tagssequences were analyzed for identity to DNA sequence in GenBank(National Center for Biotechnology Information, Bethesda, Md., USA). Therelative abundance of each transcript was determined by dividing thenumber of individual tags by total tags in the library. The copy numberof each gene was calculated assuming there are approximately 300,000transcripts in a cell (Zhang et al., 1997). The differentially expressedSAGE tags were determined by comparing the frequency of occurrence ofindividual tags in the two libraries obtained from the control (libraryLNCaP-C) and R1881 treated LNCaP cells (library LNCaP-T). The resultswere analyzed with t test, and p<0.05 was considered as a statisticallysignificant difference for a specific tag between these two libraries.

EXAMPLE 3

Kinetics of Androgen Regulation ARGs Defined by SAGE Analysis

LNCaP cells were cultured in RPMI 1640 with 10% cFBS for 5 days, thenstimulated with R1881 at 10-10, 10-8, and 10-6 M for 1, 3, 12, 24, 72,120, 168, and 216 hours. LNCaP cells identically treated but withoutR1881 served as control. The cells were harvested at indicated time andpolyA+ RNA was prepared as described as above. The polyA+ RNA wasfractionated (2 μg/lane) by running through 1% formaldehyde-agarose geland transferred to nylon membrane. The cDNA probes of several ARGs werelabeled with ³²P-dCTP by random priming (Stratagene Cloning Systems, LaJolla, Calif.). The nylon membranes were prehybridized for 2 hrs inhybridization buffer (10 mM Tris-HCl, pH 7.5, 10% Dextran sulfate, 40%Formamide, 5×SSC, 5× Denhardt's solution and 0.25 mg/ml salmon spermDNA) and hybridized to the ³²P labeled probes (1×10⁶ cpm/ml) in the samebuffer at 40° C. for 12-16 hrs. Blots were washed twice in 2×SSC/0.1%SDS for 20 min at room temperature followed by two high-stringency washwith 0.1×SSC/0.1% SDS at 50° C. for 20 min. Nylon membranes were exposedto X-ray film for autoradiography.

EXAMPLE 4

ARGs Expression Pattern in Cwr22 Model.

CWR22 (androgen dependent) and CWR22R (androgen relapsed) tumorspecimens were kindly provided by Dr. Thomas Pretlow (Case WesternReserve University School of Medicine). The tissue samples werehomogenized and polyA+ RNA was extracted with Fast Track kit(Invitrogen) following manufacture's protocol. Northern blots wereprepared as described in Example 3 and were hybridized with ³²P labeledprobes of the cDNA of interest.

Analysis of SAGE tag libraries from R1881 treated LNCaP cells. LNCaPcells were maintained in androgen deprived growth media for five daysand were treated with synthetic androgen R1881 (10 nm) for 24 hours.Since a goal of the inventors was to identify androgen signalingread-out transcripts, we chose conditions of R1881 treatment of LNCaPcells showing a robust and stable transcriptional induction ofwell-characterized prostate-specific androgen regulated genes,prostate-specific antigen (PSA) and NKX3.1 genes. A total of 90,236 tagswere derived from the two SAGE libraries. Of 90,236 tags, 6,757 tagscorresponded to linker sequences, and were excluded from furtheranalysis. The remaining 83,489 tags represented a total of 23,448 knowngenes or ESTs and 1,655 tags did not show any match in the GeneBank database. The relative abundance of the SAGE tags varied between 0.0011% and1.7%. Assuming that there are 18,000 transcripts per cell type and thereare about 83,489 anticipated total transcripts, the estimated abundanceof transcripts will be 0.2-308 copies per cell. This calculationindicated that single copy genes had high chance to be recognized bySAGE analysis in this study. The distribution of transcripts by copynumber suggests that the majority (above 90%) of the genes in ouranalysis are expressed at 1 or 2 copies level/cell. A total of 46,186and 45,309 tags were analyzed in the control (C) and R1881 (T) groupsrespectively. Unique SAGE tags corresponding to known genes, expressedsequence tags (ESTs) and novel transcripts were 15,593 and 15,920 in thecontrol and androgen treated groups respectively. About 94% of theunique SAGE tags in each group showed a match to a sequence in the genebank and 6% SAGE tags represented novel transcripts. The most abundantSAGE tags in both control and androgen treated LNCaP cells representedproteins involved in cellular translation machinery e.g., ribosomalproteins, translation regulators, mitochondrial proteins involved inbio-energetic pathways.

EXAMPLE 5

Analysis of the ARGs Defined by SAGE Tags

Of about 15,000 unique tags a total of 136 SAGE tags were significantlyup-regulated in response to R1881 whereas 215 SAGE tags weresignificantly down-regulated (p<0.05). It is important to note that of15,000 expressed sequences only 1.5% were androgen responsive suggestingthat expression of only a small subset of genes are regulated byandrogen under our experimental conditions. The ARGs identified by theinventors are anticipated to represent a hierarchy, where a fraction ofARGs are directly regulated by androgens and others represent theconsequence of the activation of direct down-stream target genes of theAR. Comparison of SAGE tags between control and R1881 also revealed that74 SAGE tags were significantly up-regulated (p<0.05) by four-fold and120 SAGE tags were significantly (p<0.05) down-regulated. Two SAGE tagscorresponding to the PSA gene sequence exhibited highest induction (16fold) between androgen treated (T) and control (C) groups. Anotherprostate specific androgen regulated gene, NKX3.1 was amongsignificantly up-regulated ARGs (8 fold). Prostate specific membraneantigen (PSMA) and Clusterin known to be down-regulated by androgenswere among the SAGE tags exhibiting decreased expression in response toandrogen (PSMA, 4 fold; Clusterin, fold). Therefore, identification ofwell characterized up-regulated and down-regulated ARGs defined by SAGEtags validates the use of LNCaP experimental model for definingphysiologically relevant ARGs in the context of prostatic epithelialcells. It is important to note that about 90% of up-regulated ARGs and98% of the down-regulated ARGs defined by our SAGE analysis were notknown to be androgen-regulated before.

EXAMPLE 6

Identification of Prostate Specific/Abundant Genes

LNCaP C/T-SAGE tag libraries were compared to a bank of 35 SAGE taglibraries (http://www.ncbi.nlm.nih.gov/SAGE/) containing 1.5 milliontags from diverse tissues and cell types. Our analysis revealed thatknown prostate specific genes e.g., PSA and NKX3.1 were found only inLNCaP SAGE tag libraries (this report and one LNCaP SAGE library presentin the SAGE tag bank). We have extended this observation to the othercandidate genes and transcripts. On the basis of abundant/uniqueexpression of the SAGE tag defined transcripts in LNCaP SAGE taglibraries relative to other libraries, we have now identified severalcandidate genes and ESTs whose expression are potentially prostatespecific or restricted (Table 4). The utility of such prostate-specificgenes includes: (a) the diagnosis and prognosis of CaP (b) tissuespecific targeting of therapeutic genes (c) candidates for immunotherapyand (d) defining prostate specific biologic functions.

Genes with defined functions showing at least five fold up ordown-regulation (p<0.05) were broadly classified on the basis of theirbiochemical function, since our results of Northern analysis ofrepresentative SAGE derived ARGs at 5-fold difference showed mostreproducible results. Table 9, presented at the end of thisspecification immediately preceding the “References” section, representsthe quantitative expression profiles of a panel of functionally definedARGs in the context of LNCaP prostate cancer cells. ARGs in thetranscription factor category include proteins involved in the generaltranscription machinery e.g., KAP1/TIF β, CHD4 and SMRT (Douarin et al.,1998; Xu et al., 1999) have been shown to participate in transcriptionalrepression. The mitochondrial transcription factor 1 (mtTF1) was inducedby 8 fold in response to R1881. A recent report describes that anothermember of the nuclear receptor superfamily, the thyroid hormonereceptor, also up-regulates a mitochondrial transcription factorexpression through a specific co-activator, PGC-1 (Wu et al., 1999). Asshown in Table 9 a thyroid hormone receptor related gene, ear-2(Miyajima et al., 1998) was also upregulated by R1881. It is striking tonote that expression of four [NKX3.1 (He et al., 1997), HOX B 13(Sreenath et al., 1999), mtTF1 and PDEF (Oettgen et al., 2000)] of theeight transcription regulators listed in Table 9 appear to be prostatetissue abundant/specific based on published reports as well as ouranalysis described above.

ARGs also include a number of proteins involved in cellular energymetabolism and it is possible that some of these enzymes may betranscriptionally regulated by mtTF1. Components of enzymes involved inoxidative decaboxylation: dihydrolipoamide succinyl transferase (Patelet al., 1995), puruvate dehydrogenase E-1 subunit (Ho et al., 1989), andthe electron tansport chain: NADH dehydrogenase 1 beta subcomplex 10(Ton et al., 1997) were upregulated by androgen. VDAC-2 (Blachly-Dysonet al., 1994), a member of small pore forming proteins of the outermitochondrial membrane and which may regulate the transport of smallmetabolites necessary for oxidative-phosphorylation, was also upregulated by androgen. Diazepam binding protein (DBI), a previousreported ARG (Swinnen et al., 1996), known to be associated with theVDAC complex and implicated in a multitude of functions includingmodulation of pheripheral benzodiaepine receptor, acyl-CoA metabolismand mitochondrial steroidogenesis (Knudsen et al., 1993) were alsoinduced by androgen in our study. A thioredoxin like protein(Miranda-Vizuete et al., 1998) which may function in modulating thecellular redox state was down regulated by androgen. In general, itappears that modulation of ARGs involved in regulating cellular redoxstatus and energy metabolism may effect reactive oxygen speciesconcentrations.

A number of cell proliferation associated proteins regulating cellcycle, signal transduction and cellular protein trafficking wereupregulated by androgen, further supporting the role of androgens insurvival and growth of prostatic epithelial cells. Androgen regulationof two proteins: XRCC2 (Cartwright et al., 1998) and RPA3 (Umbricht etal., 1993) involved in DNA repair and recombination is a novel andinteresting finding. Induction of these genes may represent a responseto DNA damage due to androgen mediated pro-oxidant shift, or these genessimply represent components of genomic surveillance mechanismsstimulated by cell proliferation. The androgen induction of a p53inducible gene, PIG 8 (Umbricht et al., 1997), is another intriguingfinding as the mouse homolog of this gene, ei24 (Gu et al., 2000), isinduced by etoposide known to generate reactive oxygen species. Inaddition, components of protein kinases modulated by adenyl cyclase,guanyl cyclase and calmodulin involved in various cellular signaltransduction stimuli were also regulated by androgen.

Gene expression modulations in RNA processing and translation componentsis consistent with increased protein synthesis expected in cells thatare switched to a highly proliferative state. Of note is nucleolin, oneof the highly androgen induced genes (12 fold) which is an abundantnucleolar protein associating with cell proliferation and plays a directrole in the biogenesis, processing and transport of ribosomes to thecytoplasm (Srivastava and Pollard, 1999). Another androgen up-regulatedgene, exportin, a component of the nuclear pore, may be involved in theshuttling of nucleolin. Androgen regulation of SiahBP1 (Page-McCaw etal., 1999), GRSF-1 (Qian and Wilusz, 1994) and PAIP1 (Craig et al.,1998) suggests a role of androgen signaling in the processing of newlytranscribed RNAs. Two splicesosomal genes, snRNP-G and snRNP-E codingfor small ribo-nucleoproteins were down-regulated by androgen. Theunr-interacting protein, UNRIP (Hunt et al., 1999) which is involved inthe direct ribosome entry of many viral and some cellular mRNAs into thetranslational pathway, was the most down-regulated gene in response toandrogen.

Quantitative evaluation of gene expression profiles by SAGE approachhave defined yeast transcriptome (Velculescu et al., 1997) and haveidentified critical genes in biochemical pathways regulated by p53(Polyak et al., 1997), x-irradiation (Hermeking et al., 1997) and theAPC gene (Korinek et al., 1997). Potential tumor biomarkers in colon(Zhang et al., 1997), lung (Hibi et al., 1998), and breast (Nacht etal., 1999) cancers and genes regulated by other cellular stimuli (Waardet al., 1999; Berg et al., 1999) have also been identified by SAGE. SAGEtechnology has enabled us to develop the first quantitative database ofandrogen regulated transcripts. Comparison of our SAGE tag libraries tothe SAGE TagBank has also revealed a number of new candidate genes andESTs whose expression is potentially abundant or specific to theprostate. We have also identified a large number of transcripts notpreviously defined as ARGs.

A great majority of functionally defined genes that were modulated byandrogen in our experimental system appear to promote cellproliferation, cell survival, gain of energy and increased oxidativereactions shift in the cells. However, a substantial fraction of theseARGs appears to be androgen specific since they do not exhibitappreciable change in their expression in other studies examining cellproliferation associated genes (Iyer et al., 1999,genome-www.stanford.edu/serum) or estrogen regulated genes in MCF7 cells(Charpentier et al., 2000). The interesting experimental observation ofRipple et al. (Ripple et al., 1997) showing a prooxidant-antioxidantshift induced by androgen in prostate cancer cells is supported by ouridentification of specific ARGs (upregulation of enzymes involved inoxidative reactions, electron transport chain and lipid metabolism inmitochondria and down regulation of thioredoxin like protein) that maybe involved in the induction of oxidative stress by androgen.

EXAMPLE 7

Characterization of the Androgen-Regulated Gene PMEPA1

cDNA library screening and Sequencing of cDNA clone. One of the SAGEtags (14 bp) showing the highest induction by androgen (29-fold)exhibited homology to an EST in the GenBank EST database. PCR primers(5′GGCAGAACACTCCGCGCTTCTTAG3′ (SEQ ID NO. 5) and5′CAAGCTCTCTTAGCTTGTGCATTC3′ (SEQ ID NO. 6)) were designed based on theEST sequence (accession number AA310984). RT-PCR was performed using RNAfrom R1881 treated LNCaP cells and the co-identity of the PCR product tothe EST was confirmed by DNA sequencing. Using the PCR product as probe,the normal prostate cDNA library was screened through the serviceprovided by Genome Systems (St. Louis, Mo.). An isolated clone, GS 22381was sequenced using the 310 Genetic Analyzer (PE Applied Biosystems,Foster Calif.) and 750 bp of DNA sequence was defined, which included2/3 of the coding region of PMEPA1. A GenBank search with PMEPA1 cDNAsequence revealed one EST clone (accession number AA088767) homologousto the 5′ region of the PMEPA1 sequence. PCR primers were designed usingthe EST clone (5′ primer) and PMEPA1 (3′ primer) sequence. cDNA fromLNCaP cells was PCR amplified and the PCR product was sequenced usingthe PCR primers. The sequences were verified using at least twodifferent primers. A contiguous sequence of 1,141 bp was generated bythese methods.

Kinetics of Androgen Regulation of PMEPA1 Expression in LNCaP Cells.

LNCaP cells (American Type Culture Collection, ATCC, Rockville Md.) weremaintained in RPMI 1640 media (Life Technologies, Inc., Gaithersburg,Md.) supplemented with 10% fetal bovine serum (FBS, Life Technologies,Inc., Gaithersburg, Md.) and experiments were performed on cellscultured between passages 20 and 30. For the studies of androgenregulation, charcoal/dextran stripped androgen-free FBS (cFBS, GeminiBio-Products, Inc., Calabasas, Calif.) was used. LNCaP cells werecultured first in RPMI 1640 with 10% cFBS for 5 days, and thenstimulated with R1881 (DUPONT, Boston, Mass.) at 10⁻¹⁰ M and 10⁻⁸ M for3, 6, 12 and 24 hours. LNCaP cells identically treated but without R1881served as control. To study the effects of androgen withdrawal on PMEPA1gene expression, LNCaP cells were cultured in RPMI 1640 with 10% cFBSfor 24, 72 and 96 hours. Poly A+ RNA samples derived from cells treatedwith or without R1881 were extracted at indicated time points with aFast Track mRNA extraction kit (Invitrogen, Carlsbad, Calif.) followingthe manufacturer's protocol. Poly A+ RNA specimens (2 zg/lane) wereelectrophoresed in a 1% formaldehyde-agarose gel and transferred to anylon membrane. Two PMEPA1 probes used for Northern blots analysis were(a) cDNA probe spanning nucleotides 3-437 of PMEPA1 cDNA sequence (SeeTable 1) and (b) 71-mer oligonucleotide between nucleotides 971 to 1,041of PMEPA1 cDNA sequence (See Table 1).

The cDNA probe was generated by RT-PCR with primers5′CTTGGGTTCGGGTGAAAGCGCC 3′ (SEQ ID NO. 7) (sense) and5′GGTGGGTGGCAGGTCGATCTCG 3′ (SEQ ID NO. 8) (antisense). PMEPA1oligonucleotide and cDNA probes and glyceraldehyde phosphatedehydrogenase gene (GAPDH) cDNA probe were labeled with ³²P-dCTP using3′ end tailing for oligonucleotides (Promega, Madison, Wis.) and randompriming for cDNA (Stratagene, La Jolla, Calif.). The nylon membraneswere treated with hybridization buffer (10 mM Tris-HCl, pH 7.5, 10%Dextran sulfate, 40% Formamide, 5×SSC, 5× Denhardt's solution and 0.25mg/ml salmon sperm DNA) for two hours followed by hybridization in thesame buffer containing the ³²P labeled probes (1×10⁶ cpm/ml) for 12-16hrs at 40° C. Blots were washed twice in 2×SSC/0.1% SDS for 20 min atroom temperature followed by two high-stringency washes with0.1×SSC/0.1% SDS at 58° C. for 20 min. Nylon membranes were exposed toX-ray film for autoradiography. The bands on films were then quantifiedwith NIH-Image processing software.

PMEPA1 expression analysis in CWR22 tumors. CWR22 is anandrogen-dependent, serially transplantable nude mouse xenograft derivedfrom a primary human prostate cancer. Transplanted CWR22 tumors arepositive for AR and the growth of CWR22 is androgen dependent. CWR22tumors regress initially upon castration followed by a relapse. Therecurrent CWR22 tumors (CWR22R) express AR, but the growth of thesetumors become androgen-independent (Gregory et al., 1998; Nagabhushan etal., 1996). One CWR22 and four CWR22R tumor specimens were kindlyprovided by Dr. Thomas Pretlow's laboratory (Case Western ReserveUniversity School of Medicine). Tumor tissues were homogenized and polyA+ RNA were extracted as above. PolyA+ RNA blots were made andhybridized as described above.

PMEPA1 expression analysis in multiple human tissues and cell lines.Multiple Tissue Northern blots containing mRNA samples from 23 humantissues and Master Dot blots containing mRNA samples from 50 differenthuman tissues were purchased from ClonTech (Palo Alto, Calif.). Theblots were hybridized with PMEPA1 cDNA and oligo probes, as describedabove. The expression of PMEPA1 in normal prostate epithelial cells(Clonetics, San Diego, Calif.), prostate cancer cells PC3 (ATCC) andLNCaP cells and breast cancer cells MCF7 (ATCC) was also analyzed bynorthern blot.

In situ hybridization of PMEPA1 in prostate tissues. A 430 bp PCRfragment (PCR sense primer: 5′CCTTCGCCCAGCGGGAGCGC 3′, (SEQ ID NO. 9)PCR antisense primer 5′CAAGCTCTCTTAGCTTGTGCATTC3′ (SEQ ID NO. 10) wasamplified from cDNA of LNCaP cells treated by R1881 and was cloned intoa PCR-blunt IITOPO vector (Invitrogen, Carlsbad, Calif.). Digoxigeninlabeled antisense and sense riboprobes were synthesized using an invitro RNA transcription kit (Boehringer Mannheim, GMbH, Germany) and alinearized plasmid with PMEPA1 gene fragment as templates. Frozen normaland malignant prostate tissues were fixed in 4% paraformaldehyde for 30min. Prehybridization and hybridization were performed at 55° C. Afterhybridization, slides were sequentially washed with 2×SSC at roomtemperature for 30 min, 2×SSC at 58° C. for 1 hr and 0.1×SSC at 58° C.for 1 hr. Antibody against digoxygenin was used to detect the signal andNBT/BCIP was used as substrate for color development (BoehringerMarnnheim, GMbH, Germany). The slides were evaluated under an OlympusBX-60 microscope. Full-length PMEPA1 coding sequence and chromosomallocalization.

Analysis of the 1,141 bp PMEPA1 cDNA sequence (SEQ ID NO.1) revealed anopen reading frame of 759 bp nucleotides (SEQ ID NO. 2) encoding a 252amino acid protein (SEQ ID NO. 3) with a predicted molecular mass of27.8 kDa, as set forth below in Table 1. TABLE 1 (SEQ ID NO. 1)TCCTTGGGTTCGGGTGAAAGCGCCTGGGGGTTCGTGGCCATGATCCCCGAGCTGCTGGAGAACTGAAGGCGGACAGTCTCCTGCGAAAC90          ▾AGGCAATGGCGGAGCTGGAGTTTGTTCAGATCATCATCATCGTGGTGGTGATGATGGTGATGGTGGTGGTGATCACGTGCCTGCTGAGCC180 (SEQ ID NO. 3)      M  A  E  L  E  F  V  Q  I  I  I  I  V  V  V  M  M  V  M  V  V  V  I  T  C  L  L  S28                                                                                   ▾ACTACAAGCTGTCTGCACGGTCCTTCATCAGCCGGCACAGCCAGGGGCGGAGGAGAGAAGATGCCCTGTCCTCAGAAGGATGCCTGTGGC270H  Y  K  L  S  A  R  S  F  I  S  R  H  S  Q  G  R  R  R  E  D  A  L  S  S  E  G  C  L  W58                                          ▾CCTCGGAGAGCACAGTGTCAGGCAACGGAATCCCAGAGCCGCAGGTCTACGCCCCGCCTCGGCCCACCGACCGCCTGGCCGTGCCGCCCT360P  S  E  S  T  V  S  G  N  G  I  P  E  P  Q  V  Y  A  P  P  R  P  T  D  R  L  A  V  P  P88TCGCCCAGCGGGAGCGCTTCCACCGCTTCCAGCCCACCTATCCGTACCTGCAGCACGAGATCGACCTGCCACCCACCATCTCGCTGTCAG450F  A  Q  R  E  R  F  H  R  F  Q  P  T  Y  P  Y  L  Q  H  E  I  D  L  P  P  T  I  S  L  S118ACGGGGAGGAGCCCCCACCCTACCAGGGCCCCTGCACCCTCCAGCTTCGGGACCCCGAGCAGCAGCTGGAACTGAACCGGGAGTCGGTGC540D  G  E  E  P  P  P  Y  Q  G  P  C  T  L  Q  L  R  D  P  E  Q  Q  L  E  L  N  R  E  S  V148GCGCACCCCCAAACAGAACCATCTTCGACAGTGACCTGATGGATAGTGCCAGGCTGGGCGGCCCCTGCCCCCCCAGCAGTAACTCGGGCA630R  A  P  P  N  R  T  I  F  D  S  D  L  M  D  S  A  R  L  G  G  P  C  P  P  S  S  N  S  G178TCAGCGCCACGTGCTACGGCAGCGGCGGGCGCATGGAGGGGCCGCCGCCCACCTACAGCGAGGTCATCGGCCACTACCCGGGGTCCTCCT720I  S  A  T  C  Y  G  S  G  G  R  M  E  G  P  P  P  T  Y  S  E  V  I  G  H  Y  P  G  S  S208TCCAGCACCAGCAGAGCAGTGGGCCGCCCTCCTTGCTGGAGGGGACCCGGCTCCACCACACACACATCGCGCCCCTAGAGAGCGCAGCCA810F  Q  H  Q  Q  S  S  G  P  P  S  L  L  E  G  T  R  L  H  H  T  H  I  A  P  L  E  S  A  A238TCTGGAGCAAAGAGAAGGATAAACAGAAAGGACACCCTCTCTAGGGTCCCCAGGGGGGCCGGGCTGGGGCTGCGTAGGTGAAAAGGCAGA900 I  W  S  K  E  K  D  K  Q  K  G  H  P  L  * 252ACACTCCGCGCTTCTTAGAAGAGGAGTGAGAGGAAGGCGGGGGGCGCAGCAACGCATCGTGTGGCCCTCCCCTCCCACCTCCCTGTGTAT990AAATATTTACATGTGATGTCTGGTCTGAATGCACAAGCTAAGAGAGCTTGCAAAAAAAAAAAGAAAAAAGAAAAAAAAAAACCACGTTTC1080                                                      ▾TTTGTTGAGCTGTGTCTTGAAGGCAAAAGAAAAAAAATTTCTACAGTAAAAAAAAAAAAAA   1141

As indicated above, Table 1 represents the nucleotide and predictedamino acid sequence of PMEPA1 (GenBank accession No. AF224278). Thepotential initiation methionine codon and the translation stop codonsare indicated in bold. The transmembrane domain is underlined. Thelocations of the intron/exon boundaries are shown with arrows, whichwere determined by comparison of the PMEPA1 cDNA sequence to thepublicly available sequences of human clones RP5-1059L7 and 718J7(GenBank accession No. AL121913 and AL035541).

A GenBank search revealed a sequence match of PMEPA1 cDNA to two genomicclones, RP5-1059L7 (accession number AL121913 in the GenBank/htgcdatabase) and 718J7 (accession number AL035541 in the GenBank/nrdatabase). These two clones mapped to Chromosome 20q13.2-13.33 andChromosome 20q13.31-13.33. This information provided evidence thatPMEPA1 is located on chromosome 20q13.

The intron/exon juctions of PMEPA1 gene were determined based on thecomparison of the sequences of PMEPA1 and the two genomic clones. Aprotein motif search using ProfileScan(http://www.ch.embnet.org/cgi-bin/TMPRED) indicated the existence of atype Ib transmembrane domain between amino acid residues 9 to 25 of thePMEPA1 sequence. Another GenBank search further revealed that the PMEPA1showed homology (67% sequence identity and 70% positives at proteinlevel) to a recently described novel cDNA located on chromosome 18(accession number NM_(—)004338) (Yoshikawa et al., 1998), as set forthbelow in Table 2. In addition to the sequence similarity, PMEPA1 alsoshares other features with C18orf1, e.g., similar size of the predictedprotein and similar transmembrane domain as the 1 isoform of C18orf1.TABLE 2 2 AELEFVQIIIIVVVMMVMVVVITCLLSHYKLSARSFISRHSQGRRREDALSSEGCLWPSE61 PMEPA1 (SEQ ID NO: 11)AELEF QIIIIVVV  V VVVITCLL+HYK+S RSFI+R +Q RRRED L  EGCLWPS+ 3AELEFAQIIIIVVVVTVMVVVIVCLLNHYKVSTRSFINRPNQSRRREDGLPQEGCLWPSD 62 C18orf1(SEQ ID NO: 12) 62STVSGNGIPEPQVYAPPRPTDRLAVPPFAQRERFHRFQPTYPYLQHEIDLPPTISLSDGE 121 PMEPA1S     G  E  +   PR  DR   P F QR+RF RFQPTYPY+QHEIDLPPTISLSDGE 63SAAPRLGASE--IMHAPRSRDRFTAPSFIQRDRFSRFQPTYPYVQHEIDLPPTISLSDGE 120 C18orf1122 EPPPYQGPCTLQLRDPEQQLELNRESVRAPPNRTIFDSDLMDSARL-GGPCPPSSNSGIS 180PMEPA1 EPPPYQGPCTLQLRDPEQQ+ELNRESVRAPPNRTIFDSDL+D A   GGPCPPSSNSGIS 121EPPPYQGPCTLQLRDPEQQMELNRESVRAPPNRTIFDSDLIDIAMYSGGPCPPSSNSGIS 180 C18orf1181 ATCYGSGGRMEGPPPTYSEVIGHYPGSSFQHQQSSGPPSLLEGTRLHHTHIAPLESAAIW 240PMEPA1 A+   S GRMEGPPPTYSEV+GH+PG+SF H Q S   +   G+RL        ES  + 181ASTCSSNGRMEGPPPTYSEVMGHHPGASFLHHQRS---NAHRGSRLQFQQ-NNAESTIVP 236 C18orf1241 SKEKDKQKGH  250 PMEPA1  K KD++ G+ 237 IKGKDRKPGN  246 C18orf1In Table 2, a “+” denotes conservative substitution.Analysis of PMEPA1 Expression

Northern hybridization revealed two transcripts of ˜2.7 kb and 5 kbusing either PMEPA1 cDNA or oligo probe. The signal intensity of bandsrepresenting these two transcripts was very similar on the X-ray filmsof the northern blots. RT-PCR analysis of RNA from LNCaP cells with fourpairs of primers covering different regions of PMEPA1 protein codingregion revealed expected size of bands from PCR reactions, suggestingthat two mRNA species on northern blot have identical sequences in theprotein coding region and may exhibit differences in 5′ and/or3′non-coding regions. However, the exact relationship between the twobands remains to be established. Analysis of multiple northern blotscontaining 23 human normal tissues revealed the highest level of PMEPA1expression in prostate tissue. Although other tissues expressed PMEPA1,their relative expression was significantly lower as compared toprostate (FIG. 1). In situ RNA hybridization analysis of PMEPA1expression in prostate tissues revealed abundant expression in theglandular epithelial compartment as compared to the stromal cells.However, both normal and tumor cells in tissue sections of primary tumortissues revealed similar levels of expression.

Androgen Dependent Expression of PMEPA1

As discussed above, PMEPA1 was originally identified as a SAGE tagshowing the highest fold induction (29-fold) by androgen. Androgendepletion of LNCaP cells resulted in decreased expression of PMEPA1.Androgen supplementation of the LNCaP cell culture media lackingandrogen caused induction of both ˜2.7 and ˜5.0 bp RNA species of PMEPA1in LNCaP cells in a dose and time dependent fashion (FIG. 2A). Basallevel of PMEPA1 expression was detected in normal prostatic epithelialcell cultures and androgen-dependent LNCaP cells cultured in regularmedium. PMEPA1 expression was not detected in AR negative CaP cells, PC3or in the breast cancer cell line, MCF7 (FIG. 2B).

Evaluation of PMEPA1 expression in androgen sensitive and androgenrefractory tumors of CWR 22 prostate cancer xenograft model

Previous studies have described increased expression of ARGs in the“hormone refractory” CWR22R variants of the CWR22 xenograft, suggestingthe activation of AR mediated cell signaling in relapsed CWR22 tumorsfollowing castration. The androgen sensitive CWR22 tumor expresseddetectable level of PMEPA1 transcripts. However, three of the fourCWR22R tumors exhibited increased PMEPA1 expression (FIG. 8).

EXAMPLE 8

Structural Features of the PMEPA1 Gene.

Analysis of a 1,141 base pair PMEPA1 cDNA sequence revealed an openreading frame of 759 nucleotides (SEQ ID NO:2) that encodes a 252 aminoacid protein (SEQ ID NO:3). A protein motif search using ProfileScan(http://www.ch.embnet.org/cAibin/TMPRED) indicated the existence of atype Ib transmembrane domain between amino acid residues 9 to 25 of thePMEPA1 sequence. In addition, the motif search revealed two PY motifs inthe PMEPA1 protein sequence, PPPY (SEQ ID NO:80) (“PY1”) and PPTY (SEQID NO:81) (“PY2”). The PY motif is a proline-rich peptide sequence witha consensus PPXY sequence (where X represents any amino acid) that canbind to proteins with WW domains [Jolliffe et al., Biochem. J., 351:557-565, 2000; Harvey et al., Trends Cell Biol., 9: 166-169, 1999;Hicke, Cell, 106: 527-530, 2001; Kumar et al., Biochem. Biophys. Res.Commun., 185: 1155-1161, 1992; Kumar et al., Genomics, 40: 435-443,1997; Sudol, Trends Biochem. Sci., 21: 161-163, 1996; Harvey et al., J.Biol. Chem., 277: 9307-9317, 2002; and Brunschwig et al., Cancer Res.,63: 1568-1575, 2003].

A protein sequence homology search revealed that PMEPA1 has an 83%sequence identity with a mouse NEDD4 WW binding protein 4 (“N4WBP4,”Accession number AK008976) (4), as shown below in Table 10. In Table 2,the + denotes a conservative substitution, and the PY motifs areunderlined. TABLE 10 Human PMEPA1: 1MAELEFVQXXXXXXXXXXXXXXXTCLLSHYKLSARSFISRHSQGRRREDALSSEGCLWPS 60 SEQ IDNO. 3 + ELEFVQ               TCLLSHYKLSARSFISRHSQ RRR+D LSSEGCLWPS MouseN4WBP4: 18 ITELEFVQIVVIVVVMMVMVVMITCLLSHYKLSARSFISRHSQARRRDDGLSSEGCLWPS77 SEQ ID NO. 68 Human PMEPA1: 61ESTVSGNGIPEPQVYAPPRPTDRLAVPPFAQRERFHRFQPTYPYLQHEIDLPPTISLSDG 120ESTVSG G+PEPQVYAPPRPTDRLAVPPF QR    RFQPTYPYLQHEI LPPTISLSDG MouseN4WBP4: 78 ESTVSG-GMPEPQVYAPPRPTDRLAVPPFIQRS---RFQPTYPYLQHEIALPPTISLSDG133 Human PMEPA1: 121EEPPPYQGPCTLQLRDPEQQLELNRESVRAPPNRTIFDSDLMDSARLGGPCPPSSNSGIS 180EEPPPYQGPCTLQLRDPEQQLELNRESVRAPPNRTIFDSDL+DS  LGGPCPPSSNSGIS MouseN4WBP4: 134 EEPPPYQGPCTLQLRDPEQQLELNRESVRAPPNRTIFDSDLIDSTMLGGPCPPSSNSGIS193 Human PMEPA1: 181ATCYGSGGRMEGPPPTYSEVIGHYPGSSFQHQQSSGPPSLLEGTRLHHTHIAPLESAAIW 240ATCY SGGRMEGPPPTYSEVIGHYPGSSFQHQQS+GP SLLEGTRLHH+HIAPLE Mouse N4WBP4:194 ATCYSSGGRMEGPPPTYSEVIGHYPGSSFQHQQSNGPSSLLEGTRLHHSHIAPLE----- 248Human PMEPA1: 241 SKEKDKQKGHPL  252 +KEK+KQKGHPL Mouse N4WBP4: 249NKEKEKQKGHPL  260

The WW domains of NEDD4 protein facilitate its binding to the targetproteins via interaction with the PY motifs of NEDD4 binding proteins[Jolliffe et al., Biochem. J., 351: 557-565, 2000; Sudol M, TrendsBiochem. Sci., 21: 161-163, 1996; Harvey et al., J. Biol. Chem., 277:9307-9317, 2002; Macias et al., Nature, 382: 646-649, 1996; Chen et al.,Proc. Natl. Acad. Sci., USA., 92: 7819-7823, 1995; and Murillas et al.,J. Biol. Chem., 277: 2897-2907, 2002]. The PMEPA1 protein sequencecomprises two PY motifs, i.e., PPPY (SEQ ID NO:80) (“PY1”) and PPTY (SEQID NO:81) (“PY2”). PY1 is in the central region of the PMEPA1 proteinand PY2 is close to the carboxyl terminus of the PMEPA1 protein (Table2). Therefore, the high protein sequence identity of PMEPA1 with N4WBP4and the presence of PY motifs indicates that PMEPA1 is the human homologof N4 WBP4 and can bind to the NEDD4 protein and other proteinscontaining a WW domain.

EXAMPLE 9

PMEPA1-PY Motifs Interact with the WW Domains of NEDD4

Plasmids. Mammalian expression vectors encoding PMEPA1-V5 and PMEPA1-GFPfusion proteins were generated by PCR amplification of the PMEPA1 openreading frame. For PMEPA1-V5-pcDNA3.1 vector the following primers wereused:

-   -   5′-GCTGCTGGAGAACTGAAGGCG-3′ (SEQ ID NO:69) and    -   5′-GTGTCCTTTCTGTTTATCCTTC-3′ (SEQ ID NO:70).

For PMEPA1-GFP-pEGFP-vector the primers used were:

-   -   5′-AAGCTTGCTGCTGGAGAACTGAAGG CG-3′ (SEQ ID NO:71) and    -   5′-GAATTCGGTGTCCTTTCTGTTTATC-3′ (SEQ ID NO:72).

The V5 tag or GFP protein was fused at the carboxyl terminus of thePMEPA1 protein. The PCR product for generating PMEPA1-V5 was insertedinto pcDNA3.1-V5-His expression vector (Invitrogen, Carlsbad, Calif.).The PCR product for generating PMEPA1-GFP was digested by HindIII andEcoRI and cloned into the same sites of pEGFP vector (Clontech, PaloAlto, Calif.). PMEPA1-PY motif mutants, in which the tyrosine residue(Y) was replaced with an alanine residue (A), were created by usingQuikChange Site-Directed Mutagenesis kit (Stratagene, La Jolla, Calif.)and using the PMEPA1-V5-pcDNA3.1 vector as a template. The plasmids ofPMEPA1-PY motif mutants are as follows: PMEPA1-PY1m-V5-pcDNA3.1, withthe first PY motif mutation (Y126A), PMEPA1-PY2m-V5-pcDNA3.1, with thesecond PY motif mutation (Y197A), and PMEPA1-PY1m/PY2m-V5-pcDNA3.1, withboth the PY motif mutations (Y126A and Y197A). The sequences of all theinserts in expression vectors were verified by DNA sequencing.

A bacterial expression plasmid of human NEDD4 gene(pNEDD4WW-GSTpGEX-2TK) encoding all four WW-domains (Accession numberXM_(—)046129) fused to glutathione S-transferase (GST-WW fusionprotein), was generated by PCR amplification of the coding region of thefour WW-domains using the primers:

-   -   5′-GCAGGATCCCAACCAGATGCTGCTTGC-3′ (SEQ ID NO:73) and    -   5′-GCAGAATTCTTTTGTAATCCCTGGAGTA-3′(SEQ ID NO:74).        Normal prostate tissue derived cDNA was used as a PCR template        and the amplified fragment was cloned into the BamHI/EcoRI sites        of pGEX-2TK (Amersham Biotech, Piscataway, N.J.). A mammalian        expression vector (NEDD4-GFP-pEGFP) encoding NEDD4-GFP fusion        protein was generated using the following primers to generate        the NEDD4 gene fragment by PCR.:    -   5′-GCAAAGCTTGTCCGGTTTGCTGGAAGC-3′ (SEQ ID NO:75) and    -   5′-GCAGAATTCCCTTTTTGTTCTTATTGGTGAC-3′ (SEQ ID NO:76).

PMEPA™ and NEDD4 Protein Binding Assays. The in vitro binding of PMEPA1and NEDD4 was assessed by GST pull-down assays. GST-WW fusion proteinwas prepared and purified with glutathione-Sepharose beads per AmershamBiotech instructions. [³⁵S]methionine labeled proteins representingPMEPA1 and its mutants were generated by in vitrotranscription/translation (TNT T7 quick coupledtranscription/translation system, Promega, Madison, Wis.). Briefly, thePMEPA1-V5-pcDNA3.1 or the three mutants (2 μg) were incubated in 40 μlof reticulocyte lysate with 40 μCi of [³⁵S]methionine for 1.5 hrs at 30°C.

[³⁵S]methionine incorporation into protein was measured and samples wereequalized on the basis of cpm. The GST-WW fusion protein bound toglutathione-Sepharose beads (5 μg) was incubated with the[³⁵S]methionine labeled lysates (12 μl) in 0.4 ml of phosphate-bufferedsaline (PBS, pH 7.4), 1 mM dithiothreitol, and protease inhibitors. Thenegative control for each [³⁵S]methionine labeled lysate represented areaction mixture with equivalent amount of the lysate incubated withglutathione-Sepharose beads without GST-WW fusion protein. After 16hours of incubation at 4° C., the beads were washed six times with PBS,resuspended in SDS-PAGE sample buffer and run on 12% SDS-PAGE gel undera reducing condition. The gels were dried and autoradiographed.

Results. The interaction of PMEPA1 and NEDD4 proteins in cells wasevaluated by a co-immunoprecipitation assay. 293 cells (human embryonalkidney cells) were co-transfected with NEDD4-GFP-pEGFP vector and one ofthe PMEPA1-V5 expression vectors encoding either wt PMEPA1-V5 or the PYmutants of PMEPA1. Thirty-six hours later the cells were collected andlysed and the lysates were immunoprecipitated with anti-GFP antibody(Clontech, Palo Alto, Calif.) following the manufacturer's protocol. Theimmunoprecipitated proteins were subjected to immunoblotting with ananti-V5 tag antibody (Invitrogen).

In vitro translated [³⁵S]Methionine-labeled PMEPA1-V5 fusion protein,with the two intact PY motifs, showed binding to the GST-WW fusionprotein (FIG. 6, lane 1). PMEPA1 with PY1 or PY2 mutations revealedsignificantly decreased binding to WW domains (FIG. 6, lane 2 and lane3). Further, PMEPA1-V5 and NEDD4-GFP fusion proteins expressed in 293cells showed strong association (FIG. 7, lane 1) and the mutant PMEPA1V5proteins having single mutation of PY1 or PY2 motif or double mutationsof both PY1 and PY2 motifs exhibited significantly reduced binding toNEDD4 (FIG. 7, lanes 2, 3, and 4). Thus both in vitro and cell culturedata reveal that PMEPA1 interacts with NEDD4 and this interactioninvolves the binding of the PMEPA1 PY motifs to WW domains. The PY2motif mutation appeared to have a greater effect on binding of PMEPA1 tothe NEDD4 WW domain.

The high protein sequence identity of PMEPA1 with N4WBP4 suggests thatPMEPA1 is the human homolog of N4 WBP4.

EXAMPLE 10

PMEPA1 Down Regulates Androgen Receptor and Affects TranscriptionalTargets of the Androgen Receptor

LNCaP cells were stably transfected with PMEPA1-GFP (PMEPA-GFP-LNCaP)and pEGFP control (pEGFP-LNCaP) expression vectors. To evaluate theeffects of exogenous PMEPA1 expression on androgen receptor in LNCaPtransfectants, cells were maintained in androgen-free media for 5 dayswhich is known to down regulate endogenous PMEPA1 expression. Androgenreceptor expression was evaluated in these cells after 5 days in theandrogen free media (time, 0 hr). Androgen receptor expression was alsoevaluated in cells replenished with 0.11 nM R1881 for different timepoints (12 hours and 24 hours) after androgen withdrawal. Western blotanalysis revealed reduced expression of androgen receptor protein inPMEPA-GFP-LNCaP cells (FIG. 4A). Decreased androgen receptor proteinlevels in PMEPA1 transfectants correlated with the reduced levels of PSAprotein, a likely consequence of the attenuation of PSA gene expressiondue to relatively low levels of androgen receptor protein. PMEPA1down-regulation of androgen receptor was further supported by results ofrelative increase of PSMA levels whose expression is normally downregulated by androgen receptor. These experiments showed that PMEPA1down regulated androgen receptor, and androgen receptor transcriptionaltargets were affected correspondingly.

Because PMEPA1 is a NEDD4 binding protein, its effects on androgenreceptor expression may involve the ubiquitin-proteasome pathway. Toshow that PMEPA1's effect on androgen receptor expression does notresult from a general or non-specific effect of the upregulation of aubiquitin protein ligase in the protein degradation pathway, weevaluated the effects of PMEPA1 on androgen receptor and the p27protein, which is known to be degraded through a ubiquitin-dependentpathway. We generated a stable PMEPA1GFP-Tet-LNCaP transfectant, inwhich the expression of PMEPA1-GFP fusion protein is regulated bytetracycline (Tet-off system, Clontech). As shown in FIG. 4B, cellscultured in the medium with tetracycline lacked PMEPA1 expression(Tet-off) but overexpressed PMEPA1 when cultured in the medium withouttetracycline. The protein level of androgen receptor decreaseddramatically in PMEPA1-overexpressing cells as compared to the relativeexpression of p27 or tubulin (FIG. 4B). Taken together, these data showthat androgen receptor is a specific target of PMEPA1.

EXAMPLE 11

Golgi Association of PMEPA1 Protein.

Our studies also revealed that PMEPA1 is a Golgi-associated protein.

Immunofluorescence Assays. Plasmids were prepared as discussed above inExample 9. The immunofluorescent assays were performed following theprocedure described by Harvey et al., J. Biol. Chem., 277: 9307-9317,2002. Briefly, stable transfectants of LNCaP cells harboringPMEPA1-GFP-pEGFP (LNCaP-PMEPA1-GFP transfectant) were grown oncoverslips for two days, fixed in 2% paraformaldehyde for 15 minutes andpermeabilized in 0.2% Triton X-100 for 2 minutes. Fixed andpermeabilized cells were incubated with anti-GM130 (recognizes acis-Golgi matrix protein) or anti-TGN38 (recognizes a protein localizingto Trans-Golgi Network, TGN) monoclonal antibodies (BD TransductionLaboratory, San Diego, Calif.) at 6.25 μg/ml for 30 minutes at roomtemperature. Cells were then washed to remove excess or non-specificallybound primary antibody followed by incubation with TRITC conjugatedanti-mouse antibody (Sigma, ST. Louis, Mo.) at 1:100 dilution for 30minutes at room temperature. The sections were mounted with fluoromount(Southern Associates, Birmingham, Ala.) and the images were processedwith a Leica fluoromicroscope and Open-Lab software (Improvision,Lexington, Mass.).

Results. PMEPA1-GFP fusion protein showed peri-nuclear localization witha Golgi-like appearance. The images of sub-cellular location of GM130, acis-Golgi protein, showed similar pattern as PMEPA1-GFP fusion protein.Superimposing the images of PMEPA1-GFP fusion protein and GM130 inLNCaP-PMEPA1-GFP transfectants confirmed the localization of PMEPA1-GFPfusion protein on cis-Golgi structure. We did not observe theco-localization of PMEPA1-GFP and TGN-38, which localizes to TGN.

The sub-cellular localization of PMEPA1 is similar to two other newlyidentified NEDD4 WW domain binding proteins, N4WBP5 and N4WBP5a, whichalso localize to the Golgi complex [Harvey et al., J. Biol. Chem., 277:9307-9317, 2002; Konstas et al., J. Biol. Chem., 277: 29406-29416,2002]. N4WBP5a sequestered the trafficking of NEDD4/NEDD4-2 therebyincreasing the activity of the epithelial sodium channel (EnaC), a knowntarget down regulated by NEDD4 [Konstas et al., J. Biol. Chem., 277:29406-29416, 2002]. As a highly androgen-regulated gene and a NEDD4binding protein, the localization of PMEPA1 on the Golgi apparatussuggests that PMEPA1 is involved in protein turn-over of androgenreceptor targets.

EXAMPLE 12

PMEPA1 Inhibits Growth of Prostate Cancer Cells.

Colony-Forming Assays. To investigate the biologic effects of PMEPA1expression in regulating cell growth and the contribution of PY motifsto such functions, we performed the colony-formation assay bytransfecting various prostate cancer cell lines with expression vectorsof the wild type PMEPA1 (“wt-PMEPA1”) and PMEPA1-PY mutants.

Prostate cancer cell lines: LNCaP, PC3, and DU145 were purchased fromATCC (Rockville, Md.) and grown in the cell culture media as describedby the supplier. The LNCaP sub-lines C4, C₄₋₂ and C₄₋₂B [Hsieh et al.,Cancer Res., 53: 2852-7, 1993; Thalmann et al., Cancer Res., 54:2577-81, 1994; and Wu et al., Int. J. Cancer, 77: 887-94, 1998] werepurchased from Urocor (Oklahoma, Okla.) and cultured in T medium (5%FBS, 80% DMEM, 20% F12, 5 ug/ml insulin, 13.65 pg/ml Triiodo-Thyronine,5 ug/ml apotransferrin, 0.244 ug/ml biotin, 25 ug/ml adenine).

Three micrograms of plasmids (PMEPA1-V5-pcDNA3.1 or vector withoutPMEPA1 insert) were transfected into the 50-70% confluent cells intriplicate in 60-mm petri dishes with Lipofectamine (Invitrogen,Carlsbad, Calif.). Tumor suppressor gene p53 (wt), and mt p53 (R175H andG245D) were also used in parallel as controls. Approximately 36 hourslater, selection with G418 at 800 μg/ml (DU145 and PC3) or 400 μg/ml(LNCaP and its sublines) was initiated. Cells were maintained withG418-containing medium that was changed every 3-4 days. After 2-4 weeksof selection, the cells were rinsed with 1×PBS, fixed with 2%formaldehyde in 1×PBS for 15 minutes, stained with 0.5% crystal violetin 1×PBS for 15 minutes, and rinsed 1-2 times with distilled H₂O.Colonies visible in each dish without magnification were counted byOpen-Lab software.

To assess the effects of the PY motif mutations on the colony-formingability of PMEPA1, LNCaP and PC3 cells were also transfected with PMEPA1mutants: PMEPA1-PY1m-pcDNA3.1, PMEPA1-PY2m-pcDNA3.1, or PMEPA1-PY1m/PY2m-pcDNA3.1. PMEPA1-V5-pcDNA3.1 and expression vector without insertserved as positive and negative controls, respectively, for the PMEPA1mutants. Two independent colony-forming assays were performed as above.

As shown in FIGS. 3A-F, the colony-forming abilities of prostate cancercell lines DU145, PC3, LNCaP, and LNCaP sublines were significantlysuppressed by transfection of the sense version of the wt-PMEPA1expression vector. Under these conditions wt-p53 showed similar cellgrowth inhibition (data not shown).

In two independent experiments, mutation of the PY1 motif appears toabolish the inhibition of colony formation by wt-PMEPA1, emphasizing therole of the PY1 motif in PMEPA1 and NEDD4 interactions and the biologicfunctions of PMEPA1 (FIG. 3G-H). The growth inhibitory effect of PMEPA1appears to be linked to the interactions of PY1 motif to NEDD4 WWdomain. This interpretation is based on the striking observationsshowing distinctively more colonies with PY1 motif mutant in comparisonto wt-PMEPA1.

Cell Proliferation Analysis. To further evaluate the growth inhibitoryeffects of PMEPA1 on prostate cancer cells, a stable PMEPA1-GFP-TetLNCaP transfectant was generated. Expression of PMEPA1-GFP fusionprotein in these cells was negatively regulated by tetracycline in themedium (Clontech). For cell proliferation assays, three thousandPMEPA1-GFP-Tet LNCaP cells were seeded in 96-well plates with or without1 kg/ml of tetracycline in the medium. CellTiter 96 Aqueous One Solutionkit (Promega, Madison, Wis.) was used to measure the cell proliferationaccording to the manufacturer's instructions.

The growth inhibitory effect of PMEPA1 has been further confirmed by thecell proliferation characteristics of stable PMEPA1-GFP-Tet-LNCaP cells,where exogenous PMEPA1 is upregulated in the absence of tetracycline.The growth of the PMEPA1-GFP-Tet LNCaP cells in tetracycline negativemedium is significantly slower than that of PMEPA1-tet LNCaPtransfectant in tetracycline positive medium (FIG. 5). LNCaP cells withPMEPA1 overexpression also revealed increased RB phosphorylation furtherconfirming the cell growth inhibitory effect of PMEPA1 (data not shown).

PMEPA1 is expressed in androgen receptor positive prostate cancer celllines, including LNCaP and its sublines (C4, C₄₋₂ and C₄₋₂B). LNCaPcells are androgen dependent for growth. Even though the growth of LNCaPsublines is androgen independent, androgen receptor is critical fortheir proliferation [Zegarra-Moro et al., Cancer Res., 62: 1008-1013,2002]. We observed that overexpression of PMEPA1 by transfecting thePMEPA1 expression vector into LNCaP and its sublines significantlyinhibited the cell proliferation. Since our preliminary observationsshowed that PMEPA1 overexpression in LNCaP cells resulted in alteredexpression of androgen receptor downstream genes (Xu et al. unpublisheddata), we hypothesized that the growth inhibitory effect of PMEPA1 onLNCaP and its sublines may be mediated directly or indirectly throughaffecting androgen receptor functions. Despite the growth inhibitoryeffect on androgen receptor positive prostate cancer cell lines, PMEPA1was also found to inhibit the growth of androgen receptor negativeprostate tumor cells, DU145 and PC3, suggesting that the growthinhibitory effects of PMEPA1 on DU145 and PC3 could be mediated throughalternative mechanisms, e.g., regulation of other nuclear steroidreceptors by PMEPA1. Nonetheless, inhibition of prostate cancer cellgrowth by PMEPA1 implicates PMEPA1 in control of prostate cancerdevelopment.

EXAMPLE 13

Decreased PMEPA1 Expression in Prostate Tumor Tissues.

We also evaluated the relationship of alterations in PMEPA1 expressionto the clinico-pathologic features of prostate cancer.

Prostate Tissue Specimens, Laser Capture Microdissection (LCM) andQuantitative RT-PCR (QRT-PCR) Assay. Matched prostate cancer and normaltissues were derived from radical prostatectomy specimens from 62 CaPpatients treated at Walter Reed Army Medical Center (under anIRB-approved protocol). The procedures of collecting specimens werepreviously described [Xu et al., Cancer Res. 60: 6568-6572, 2000]. Tenmicron frozen sections were prepared and stored at −70° C.Histologically normal prostate epithelial cells and prostate tumor cellsfrom each patient were harvested using LCM equipment according to theprotocol provided by the manufacturer (Arcturus Engineering, MountainView, Calif.).

Total RNA was prepared from the harvested normal and tumor prostateepithelial cells as previously described [Xu et al., Cancer Res. 60:6568-6572, 2000] and quantified with Fluorometer (Bio-Rad, Hercules,Calif.). QRT-PCR was conducted using 0.1 ng of total RNA from pairednormal and tumor cells. PMEPA1 PCR primers were carefully designed thatonly amplify PMEPA1 but not STAG1, an alternatively spliced form ofPMEPA1 [Rae et al., Mol. Carcinog., 32: 44-53, 2001]. The PCR primerswere:

-   -   5′-CATGATCCCCGAGCTGCT-3′ (SEQ ID NO:77) and    -   5′-TGATCTGAACAAACTCCAGCTCC-3′ (SEQ ID NO:78), and the labeled        probe was:        -   5′-AGGCGGACAGTCTCCTGCGAAAC-3′ (SEQ ID NO:79).

GAPDH gene expression was detected as the internal control (PE AppliedBiosystems, Foster, Calif.). Paired triplicate samples (one lacking RTand duplicate with RT) were amplified in 50 μl volumes containing themanufacturer's recommended universal reagent, proper primers and probeof PMEPA1 or GAPDH using 7700 sequence detection system (PE AppliedBiosystems, Foster, Calif.).

Results were plotted as average cycle threshold (cT) values for eachduplicate sample minus the average duplicate cT values for GAPDH.Differences between matched tumor (T) and normal (N) samples werecalculated using 2exp(cT_(tumor)−cT_(normal)) and expressed as foldchanges in expression. The expression status of PMEPA1 was furthercategorized as either: 1) overexpression in tumor tissue (T>N), definedas 1+(1.5-3 fold), 2+(3.1-10 fold), 3+(10.1-20 fold) and 4+(>20 fold)increased expression as compared with matched normal tissue; 2) reducedexpression in tumor tissue (T<N), defined as 1—(1.5-3 fold), 2—(3.1-10fold), 3—(10.1-20 fold) and 4—(>20 fold) decreased expression ascompared with matched normal tissue; or 3) no change (T=N), defined as 0(<1.5 fold). No detectable PMEPA1 expression in one of the specimens oftumor/normal pairs was scored as 4+for increased or 4—for decreasedexpression.

Statistical analysis was performed with the SPSS software package. Theassociation between PMEPA1 expression and clinico-pathological featureswas analyzed using chi-square tests. The Kaplan-Meier curves wereapplied to display the PSA-recurrence-free survival data. A p value<0.05was considered as statistically significant.

The overall expression pattern of PMEPA1 primary prostate cancer isshown below in Table 11. TABLE 11 Number of Degree of PMEPA1 PMEPA1Patients/ Expression Expression Group (%) Quantity Number (%) T < N 40(64.5) 1− 11 (27.5) 2− 17 (42.5) 3−  5 (12.5) 4−  7 (17.5) T > N 10(16.1) 1+  6 (60.0) 2+  4 (40.0) 3+ 4+ T = N 12 (19.4) 0  

Comparison of PMEPA1 expression between tumor and normal cells revealedtumor cell associated decreased expression (T<N) in 64.5% tumorspecimens (40 of 62), increased expression (T>N) in 16.1% specimens (10of 62) and no change (T=N) in 19.4% specimens (12 of 62). When theseexpression patterns were stratified by organ-confined (pT2) andnon-organ-confined (pT3) disease, a higher percentage of PMEPA1reduction was seen in pT3 (74%) vs. pT2 (48%). Because the T>N group hasa small number of cases, we combined the T>N group and the T=N group(T>N group). As shown below in Table 12, comparison of theclinico-pathologic parameters between the T<N group and the T>N grouprevealed that the T<N group had a significantly higher percentage ofpatients with pT3 tumors (p=0.035) and more patients in this group had ahigher level of preoperative serum prostate specific antigen (PSA)(p=0.023). TABLE 12 Time to Pathologic PSA Recurrence Stage PSA Range(%) Recurrence after Surgery PMEPA1 (%) ≦4 4.1-10 10.1-20 (%) (month)Expression T2 T3 ng/ml ng/ml ng/ml No Yes Mean ± SE T < N 11 29 1 30 929 11 8.2 ± (27.5) (72.5) (2.5)  (75.0) (22.5) (72.5) (27.5) 3.4 T ≧ N12 10 5 15 2 19 3 18.4 ± (54.5) (45.5) (22.7) (68.2) (9.1)  (86.4)(13.6) 6.3 pValue 0.035 0.023 0.211 0.18

Out of 62 patients whose tumors were analyzed for PMEPA1 expression, 14patients showed prostate cancer recurrence as defined by serum PSA levelequal or higher than 0.2 ng/ml after prostatectomy. Of the 14 patients,11 showed reduced tumor associated PMEPA1 expression (78.5%). ReducedPMEPA1 expression seems to associate with a higher recurrence rate and ashorter duration to recurrence after surgery, even through thestatistical analysis did not reveal a significant difference. Theabsence of a significant difference might be due to the small number ofpatients.

The specification is most thoroughly understood in light of theteachings of the references cited within the specification which arehereby incorporated by reference. The embodiments within thespecification provide an illustration of embodiments of the inventionand should not be construed to limit the scope of the invention. Theskilled artisan readily recognizes that many other embodiments areencompassed by the invention. TABLE 3 Genes Regulated by Androgen: SAGEData Derived from CPDR SAGE Library Accession Description Effect ofAndrogen AA310984 EST Up-regulated by Androgen M26663

prostate-specific antigen mRNA, Up-regulated by Androgen complete cds.*AA508573 Human nucleolin gene, complete cds Up-regulated by AndrogenAB020637 Homo sapiens mRNA for KIAA0830 protein, partial Up-regulated byAndrogen cds. AA280663 EST Up-regulated by Androgen U31657KRAB-associated protein 1 Up-regulated by Androgen AI879709 ESTUp-regulated by Androgen AA602190 EST Up-regulated by Androgen AF035587Homo sapiens X-ray repair cross-complementing Up-regulated by Androgenprotein 2 (XRCC2) AF151898 Homo sapiens CGI-140 protein mRNAUp-regulated by Androgen AA418786 No reliable matches, only see in twolinberary (1 Up-regulated by Androgen each) AI308812 EST Up-regulated byAndrogen X59408 Membrane cofactor protein (CD46, trophoblast-Up-regulated by Androgen lymphocyte cross-reactive antigen) X81817Accessory proteins BAP31/BAP29 Up-regulated by Androgen AF071538

Ets transcription factor PDEF Up-regulated by Androgen (PDEF) mRNA,complete NM_003201 Transcription factor 6-like 1 (mitochondrialUp-regulated by Androgen transcription factor 1-like) U41387 Human Guprotein mRNA, partial cds. Up-regulated by Androgen U58855 Guanylatecyclase 1, soluble, alpha 3 Up-regulated by Androgen X12794 Human v-erbArelated ear-2 gene. Up-regulated by Androgen U88542

homeobox protein Nkx3.1 Up-regulated by Androgen D89729 Homo sapiensmRNA for CRM1 protein, complete Up-regulated by Androgen cds. U75329TMPRSS2 Up-regulated by Androgen AA062976 EST Up-regulated by AndrogenL12168 Homo sapiens adenylyl cyclase-associated protein Up-regulated byAndrogen (CAP) mRNA AA043945 EST Up-regulated by Androgen AF026291 Homosapiens chaperonin containing t-complex Up-regulated by Androgenpolypeptide 1, delta AB002301 Human mRNA for KIAA0303 gene, partial cds.Up-regulated by Androgen D13643 Human mRNA for KIAA0018 gene, completecds. Up-regulated by Androgen AI310341 EST Up-regulated by AndrogenU49436 Human translation initiation factor 5 (eIF5) mRNA, Up-regulatedby Androgen complete cds S79862 Proteasome (prosome, macropain) 26Ssubunit, non- Up-regulated by Androgen ATPase, 5 M14200 Human diazepambinding inhibitor (DBI) mRNA, Up-regulated by Androgen complete cds.AA653318 FK506-binding protein 5 Up-regulated by Androgen L07493 Homosapiens replication protein A 14 kDa subunit Up-regulated by Androgen(RPA) mRNA, AJ011916 Homo sapiens mRNA for hypothetical protein.Up-regulated by Androgen AA130537 EST Up-regulated by Androgen D16373Human mRNA for dihydrolipoamide Up-regulated by Androgensuccinyltransferase, complete cds. AL096857 Novel human mRNA fromchromosome 1 Up-regulated by Androgen AF007157 Homo sapiens clone 23856unknown mRNA, partial Up-regulated by Androgen cds. AA425929 NADHdehydrogenase (ubiquinone) 1 beta Up-regulated by Androgen subcomplex,10 (22 kD, PDSW) AI357815 EST Up-regulated by Androgen D83778 Human mRNAfor KIAA0194 gene, partial cds. Up-regulated by Androgen AF000979 Homosapiens testis-specific Basic Protein Y 1 Up-regulated by Androgen(BPY1) mRNA, AA889510 EST Up-regulated by Androgen AB018330 Homo sapiensmRNA for KIAA0787 protein, partial Up-regulated by Androgen cds.AA026941 EST Up-regulated by Androgen AA532377 Chromosome 1 open readingframe 8 Up-regulated by Androgen AF010313 Homo sapiens Pig8 (PIG8) mRNA(etoposide- Up-regulated by Androgen induced mRNA), complete cds. L06328Human voltage-dependent anion channel isoform 2 Up-regulated by Androgen(VDAC) mRNA, U41804 Human putative T1/ST2 receptor binding proteinUp-regulated by Androgen precursor mRNA, AB020676 Homo sapiens mRNA forKIAA0869 protein, partial Up-regulated by Androgen cds. J03503 Humanpyruvate dehydrogenase E1-alpha subunit Up-regulated by Androgen mRNA,cds. AA421098 EST Up-regulated by Androgen AF072836 Sox-liketranscriptional factor Up-regulated by Androgen AA115355 ESTUp-regulated by Androgen AF118240 Homo sapiens, peroxisomal biogenesisfactor 16 Up-regulated by Androgen (PEX16) mRNA, complete AA011178 ESTUp-regulated by Androgen X15573 Human liver-type 1-phosphofructokinase(PFKL) Up-regulated by Androgen mRNA, complete cds. AA120930 ESTUp-regulated by Androgen AB002321 Human mRNA for KIAA0323 gene, partialcds Up-regulated by Androgen AF151837 Homo sapiens CGI-79 protein mRNA,complete cds Up-regulated by Androgen AA481027 EST Up-regulated byAndrogen AA039343 EST Up-regulated by Androgen U09716 Humanmannose-specific lectin (MR60) mRNA, Up-regulated by Androgen completecds. AF044773 Homo sapiens breakpoint cluster region protein 1Up-regulated by Androgen (BCRG1) mRNA U51586 Human siah binding protein1 (SiahBP1) mRNA, Up-regulated by Androgen partial cds. M36341 HumanADP-ribosylation factor 4 (ARF4) mRNA, Up-regulated by Androgen completecds. AI282096 EST Up-regulated by Androgen W45510 RAB7, member RASoncogene family-like 1 Up-regulated by Androgen X16135 Human mRNA fornovel heterogeneous nuclear RNP Up-regulated by Androgen protein, Lprotein AF052134 Homo sapiens clone 23585 mRNA sequence, Up-regulated byAndrogen AF052134 D26068 Williams-Beuren syndrome chromosome region 1Up-regulated by Androgen X69433 H. sapiens mRNA for mitochondrialisocitrate Up-regulated by Androgen dehydrogenase (NADP+). X61123 B-celltranslocation gene 1, anti-proliferative Up-regulated by Androgen X63423H. sapiens mRNA for delta-subunit of mitochondrial Up-regulated byAndrogen F1F0 ATP-synthase AJ010025 Homo sapiens mRNA forunr-interacting protein. Down-regulated by Androgen AF003938 Homosapiens thioredoxin-like protein mRNA, Down-regulated by Androgencomplete cds. AB014536 Homo sapiens copine III (CPNE3) mRNADown-regulated by Androgen AA504468 EST Down-regulated by AndrogenNM_001273 Chromodomain helicase DNA binding protein 4 Down-regulated byAndrogen AA015746 Homo sapiens mRNA; cDNA DKFZp586H0722 Down-regulatedby Androgen (from clone DKFZp586H0722) AA552354 EST Down-regulated byAndrogen AA025744 3-prime-phosphoadenosine 5-prime-phosphosulfateDown-regulated by Androgen synthase 2 X71129 H. sapiens mRNA forelectron transfer flavoprotein Down-regulated by Androgen beta subunitAA046050 EST Down-regulated by Androgen U57052 Human Hoxb-13 mRNA,complete cds Down-regulated by Androgen AA400137 EST Down-regulated byAndrogen AA487586 EST Down-regulated by Androgen J04208 Humaninosine-5′-monophosphate dehydrogenase Down-regulated by Androgen (IMP)mRNA M64722 Testosterone-repressed prostate message 2 Down-regulated byAndrogen (apolipoprotein J) AI743483 EST Down-regulated by AndrogenAA476914 EST Down-regulated by Androgen AA026691 EST Down-regulated byAndrogen AI014986 EST Down-regulated by Androgen X85373 Small nuclearribonucleoprotein polypeptide G Down-regulated by Androgen U07231 G-richRNA sequence binding factor 1 Down-regulated by Androgen T97753 Glycogensynthase 2 (liver) Down-regulated by Androgen AA234050 ESTDown-regulated by Androgen AI015143 EST Down-regulated by AndrogenU09196 Human 1.1 kb mRNA upregulated in retinoic acid Down-regulated byAndrogen treated HL-60 neutrophilic cells. AA977749 EST Down-regulatedby Androgen NM_006451 Polyadenylate binding protein-interacting protein1 Down-regulated by Androgen AI818296 EST Down-regulated by AndrogenAI250561 EST Down-regulated by Androgen AA063613 EST Down-regulated byAndrogen U59209 Hs.183596: UDP glycosyltransferase 2 family,Down-regulated by Androgen polypeptide B17, U59209 Z11559Iron-responsive element binding protein 1 Down-regulated by AndrogenAF052578 Homo sapiens androgen receptor associated proteinDown-regulated by Androgen 24 (ARA24) X16312 Human mRNA forphosvitin/casein kinase II beta Down-regulated by Androgen subunit.H17890 PCTAIRE protein kinase 3 Down-regulated by Androgen AA192312 ESTDown-regulated by Androgen AA043787 EST Down-regulated by AndrogenAI052020 EST Down-regulated by Androgen AB014512 Homo sapiens mRNA forKIAA0612 protein Down-regulated by Androgen NM_001328 Homo sapiensC-terminal binding protein 1 (CTBP1) Down-regulated by Androgen mRNAM15919 Human autoimmune antigen small nuclear Down-regulated by Androgenribonucleoprotein E mRNA. AF151813 Homo sapiens CGI-55 protein mRNA,complete cds Down-regulated by Androgen L41351 Protease, serine, 8(prostasin) Down-regulated by Androgen AF077046 Homo sapiens gangliosideexpression factor 2 (GEF- Down-regulated by Androgen 2) homolog U15008Small nuclear ribonucleoprotein D2 polypeptide Down-regulated byAndrogen (16.5 kD), AA938995 N62491 Folate hydrolase (prostate-specificmembrane Down-regulated by Androgen antigen) 1 AI569591 ESTDown-regulated by Androgen AJ131245 Secretory protein 24 (SEC24).Down-regulated by Androgen U90543 Human butyrophilin (BTF1) mRNA,complete cds. Down-regulated by Androgen Z47087 Transcription elongationfactor B (SIII), polypeptide Down-regulated by Androgen 1-like M34539FK506-binding protein 1A (12 kD) Down-regulated by Androgen N43807yy19a05.r1 Soares melanocyte 2NbHM Homo Down-regulated by Androgensapiens cDNA clone U03269 Human actin capping protein alpha subunit(CapZ) Down-regulated by Androgen mRNA, complete AI571685 ESTDown-regulated by Androgen AA010412 EST Down-regulated by AndrogenL40403 Homo sapiens (clone zap3) mRNA, 3′ end of cds. Down-regulated byAndrogen NM_006560 CUG triplet repeat, RNA-binding protein 1Down-regulated by Androgen NM_004713 Serologically defined colon cancerantigen 1 Down-regulated by Androgen U36188Clathrin-associated/assembly/adaptor protein, Down-regulated by Androgenmedium 1 AB020721 KIAA0914 gene product Down-regulated by AndrogenT35365 EST Down-regulated by Androgen AF029789 Homo sapiensGTPase-activating protein (SIPA1) Down-regulated by Androgen mRNA,complete cds. AA427857 EST Down-regulated by Androgen AA910404 ESTDown-regulated by Androgen L42379 Quiescin Q6 (bone-derived growthfactor) Down-regulated by Androgen AL117641 cDNA DKFZp434L235Down-regulated by Androgen AI688119 EST Down-regulated by AndrogenAA688073 EST Down-regulated by Androgen NM_002945 Replication protein A1(70 kD) Down-regulated by Androgen AI797610 EST Down-regulated byAndrogen AF086095 Homo sapiens full length insert cDNA cloneDown-regulated by Androgen YZ88A07. AF070666 Homo sapiens tissue-typepituitary Kruppel- Down-regulated by Androgen associated box proteinR55128 Proteasome (prosome, macropain) 26 S subunit, non- Down-regulatedby Androgen ATPase, 2 X75621 Tuberous sclerosis 2 Down-regulated byAndrogen AA019070 EST Down-regulated by Androgen AI089867 ESTDown-regulated by Androgen NM_001003 Homo sapiens ribosomal protein,large, P1 (RPLP1) Down-regulated by Androgen mRNA L05093 Ribosomalprotein L18a Down-regulated by Androgen AA854176 EST Down-regulated byAndrogen AI929622 Homo sapiens clone 23675 mRNA sequence Down-regulatedby Androgen AI264769 ESTs, Weakly similar to ORF YDL087c Down-regulatedby Androgen [S. cerevisiae] L09159 Ras homolog gene family, member A,may be Down-regulated by Androgen androgen regulated? AI143187 ESTDown-regulated by Androgen H17900 cDNA DKFZp586H051 (from cloneDown-regulated by Androgen DKFZp586H051) NM_005617 Ribosomal protein S14Down-regulated by Androgen L49506 Cyclin G2 Down-regulated by AndrogenAA614448 Regulator of G-protein signalling 5 Down-regulated by AndrogenS83390 T3 receptor-associating cofactor-1 Down-regulated by AndrogenAA917672 EST Down-regulated by Androgen X52151 Arylsulphatase ADown-regulated by Androgen U09646 Carnitine palmitoyltransferase IIDown-regulated by Androgen Z50853 ATP-dependent protease ClpAP (E.coli), proteolytic Down-regulated by Androgen subunit, human AB023208MLL septin-like fusion Down-regulated by Androgen U92014 Human clone121711 defective mariner transposon Down-regulated by Androgen Hsmar2mRNA AA878293 Alpha-1-antichymotrypsin Down-regulated by AndrogenAA554191 EST Down-regulated by Androgen M55618 Hexabrachion (tenascin C,cytotactin) Down-regulated by Androgen AA027050 EST Down-regulated byAndrogen AF112472 Homo sapiens calcium/calmodulin-dependent proteinDown-regulated by Androgen kinase II beta AA583866 EST Down-regulated byAndrogen AA115687 EST Down-regulated by Androgen AA043318 ESTDown-regulated by Androgen U90329 Poly(rC)-binding protein 2Down-regulated by Androgen Y00815 Protein tyrosine phosphatase, receptortype, F Down-regulated by Androgen X76013 H. sapiens QRSHs mRNA forglutaminyl-tRNA Down-regulated by Androgen synthetase. X75861 Testisenhanced gene transcript Down-regulated by Androgen AA593078 Homosapiens PAC clone DJ0167F23 from 7p15 Down-regulated by Androgen J04058Human electron transfer flavoprotein alpha-subunit Down-regulated byAndrogen mRNA AF026292 Homo sapiens chaperonin containing t-complexDown-regulated by Androgen polypeptide 1, eta AF068754 Homo sapiens heatshock factor binding protein 1 Down-regulated by Androgen HSBP1 mRNA,NM_000172 Guanine nucleotide binding protein (G protein), Down-regulatedby Androgen alpha transducing activity polypeptide 1 AI140631 Hs.1915:folate hydrolase (prostate-specific Down-regulated by Androgen membraneantigen) 1Bold font indicates known androgen-regulated gene based on MedlineSearch.

TABLE 4 Potential Prostate Specific/Abundant Genes Derived From NCBI andCPDR SAGE Libraries Accession Description M88700 Human dopadecarboxylase (DDC) gene, complete cds. W45526 zc26b04.r1Soares_senescent_fibroblasts_NbHSF Homo sapiens cDNA, Hs.108981: ficolin(collagen/fibrinogen domain-containing) 1, AF201077 NADH: ubiquinoneoxidoreductase MLRQ subunit (NDUFA4) mRNA, complete cds with polyA.D55953 HUM407H12B Clontech human fetal brain polyA + mRNA (#6535) Homo,Hs.118724: histidine triad nucleotide-binding protein, AJ012499, mRNAactivated in tumor suppression, clone TSAP19 with polyA AA082804zn41g02.r1 Stratagene endothelial cell 937223 Homo sapiens cDNA,Hs.110967: ESTs, Weakly similar to KIAA0762 protein [H. sapiens],Hs.5662: guanine nucleotide binding protein (G protein), betapolypeptide 2-like 1 in the sequence no tag X05332 Human mRNA forprostate specific antigen.* AI278854 qo42f01.x1 NCI_CGAP_Lu5 Homosapiens cDNA clone IMAGE: 1911193 3′, NM_004537, nucleosome assemblyprotein 1-like 1 (NAP1L1), tag is at beginning of the gene. W75950zd58b02.r1 Soares_fetal_heart_NbHH19W Homo sapiens cDNA clone, AF151840,CGI- 82 protein mRNA, tag is at 3′ end. F02980 HSC1IC062 normalizedinfant brain cDNA Homo sapiens cDNA clone M99487 Human prostate-specificmembrane antigen (PSM) mRNA, complete cds. AL035304 H. sapiens gene fromPAC 295C6, similar to rat PO44. AI088979 ou86f03.s1Soares_NSF_F8_9W_OT_PA_P_S1 Homo sapiens cDNA clone AF186249

six transmembrane epithelial antigen of prostate (STEAP1) mRNA C15801C15801 Clontech human aorta polyA + mRNA (#6572) Homo sapiens cDNAL10340 Human elongation factor-1 alpha (ef-1) mRNA, 3′ end. NM_004540Homo sapiens neural cell adhesion molecule 2 (NCAM2) AA151796 zl39c02.r1Soares_pregnant_uterus_NbHPU Homo sapiens cDNA clone NM_001634 Homosapiens S-adenosylmethionine decarboxylase 1 (AMD1) NM_005013 Homosapiens nucleobindin 2 (NUCB2)AL121913 in GenBank htgc database) and718J7 (Accession number AL035541 AF004828 Homo sapiens rab3-GAPregulatory domain mRNA, complete cds. X60819 X60 H. sapiens DNA formonoamine oxidase type A (14) (partial). AA133972 zl38g12.r1Soares_pregnant_uterus_NbHPU Homo sapiens cDNA clone M69226 Humanmonoamine oxidase (MAOA) mRNA, complete cds. AA969141 op50c11.s1Soares_NFL_T_GBC_S1 Homo sapiens cDNA clone AA523652 ni64d09.s1NCI_CGAP_Pr12 Homo sapiens cDNA clone IMAGE: 981617, mRNA AF078749 Homosapiens organic cation transporter 3 (SLC22A3) AA583544 nf25h10.s1NCI_CGAP_Pr1 Homo sapiens cDNA clone IMAGE: 914851, mRNA AF051894 Homosapiens 15 kDa selenoprotein mRNA, complete cds. AF165967 Homo sapiensDDP-like protein mRNA X57129 H. sapiens H1.2 gene for histone H1.AA640928 nr28d08.r1 NCI_CGAP_Pr3 Homo sapiens cDNA clone IMAGE: 1169295,mRNA U41766 Human metalloprotease/disintegrin/cysteine-rich proteinprecursor AF023676 Homo sapiens lamin B receptor homolog TM7SF2 (TM7SF2)mRNA, U10691 Human MAGE-6 antigen (MAGE6) gene, complete cds. M22976Human cytochrome b5 mRNA, 3′ end. L14778 Human calmodulin-dependentprotein phosphatase catalytic subunit AF071538

Ets transcription factor PDEF (PDEF) mRNA, complete U39840 Humanhepatocyte nuclear factor-3 alpha (HNF-3 alpha) mRNA, AA532511nj54d03.s1 NCI_CGAP_Pr9 Homo sapiens cDNA clone IMAGE: 996293, mRNAX07166 Human mRNA for enkephalinase (EC 3.4.24.11). M96684 H. sapiensPur (pur-alpha) mRNA, complete cds. AI204040 qe77f05.x1Soares_fetal_lung_NbHL19W Homo sapiens cDNA clone AA577923 nl20a01.s1NCI_CGAP_HSC1 Homo sapiens cDNA clone IMAGE: 1041192, AA569633nm38h09.s1 NCI_CGAP_Pr4.1 Homo sapiens cDNA clone IMAGE: 1062497, U65011Human preferentially expressed antigen of melanoma (PRAME) mRNA, U21910Human basic transcription factor BTF2p44 mRNA, 3′ end, partial cds.AA633187 nq07c12.s1 NCI_CGAP_Lu1 Homo sapiens cDNA clone IMAGE: 11431903′ AF000993 Homo sapiens ubiquitous TPR motif, X isoform (UTX) mRNA,W76105 zd65b04.r1 Soares_fetal_heart_NbHH19W Homo sapiens cDNA cloneH39906 yo54a07.r1 Soares breast 3NbHBst Homo sapiens cDNA clone AA971717op95c11.s1 NCI_CGAP_Lu5 Homo sapiens cDNA clone IMAGE: 1584596 3′,M68891 Human GATA-binding protein (GATA2) mRNA, complete cds. AA310157EST181013 Jurkat T-cells V Homo sapiens cDNA 5′ end, mRNA sequence.X00948 Human mRNA for prepro-relaxin H2. AB018330 Homo sapiens mRNA forKIAA0787 protein, partial cds. AA890637 ak11e11.s1Soares_parathyroid_tumor_NbHPA Homo sapiens cDNA clone M64929 J05 Humanprotein phosphatase 2A alpha subunit mRNA, complete cds. W24341zb81h12.r1 Soares_senescent_fibroblasts_NbHSF Homo sapiens cDNA AA974479od58b03.s1 NCI_CGAP_GCB1 Homo sapiens cDNA clone IMAGE: 1372109 3′R31644 yh69e05.r1 Soares placenta Nb2HP Homo sapiens cDNA clone AA573246nm52c02.s1 NCI_CGAP_Br2 Homo sapiens cDNA clone IMAGE: 1071842 3′,AA507635 ng84b02.s1 NCI_CGAP_Pr6 Homo sapiens cDNA clone IMAGE: 941451,mRNA gb|AF008915 Homo sapiens EVI5 homolog mRNA AL049987 Homo sapiensmRNA; cDNA DKFZp564F112 (from clone DKFZp564F112). U81599

homeodomain protein HOXB13 mRNA AA641596 nr20f05.s1 NCI_CGAP_Pr2 Homosapiens cDNA clone IMAGE: 1168545, mRNA D84295 Human mRNA for possibleprotein TPRDII R13859 yf65d08.r1 Soares infant brain 1NIB Homo sapienscDNA clone M34840 Human prostatic acid phosphatase mRNA, complete cds.AA572913 nm42f12.s1 NCI_CGAP_Pr4.1 Homo sapiens cDNA clone IMAGE:1062863, AA094460 cp0378.seq.F Human fetal heart, Lambda ZAP ExpressHomo sapiens AF031166 Homo sapiens SRp46 splicing factor retropseudogenemRNA. AA625147 af70c07.r1 Soares_NhHMPu_S1 Homo sapiens cDNA cloneIMAGE: 1047372 T39510 ya06h07.r1 Stratagene placenta (#937225) Homosapiens cDNA clone R35034 yg60h03.r1 Soares infant brain 1NIB Homosapiens cDNA clone AI003674 zg01c04.s1 Soares_pineal_gland_N3HPG Homosapiens cDNA clone AJ003636 AJ003636 Selected chromosome 21 cDNA libraryHomo sapiens cDNA AA601385 no16d12.s1 NCI_CGAP_Phe1 Homo sapiens cDNAclone IMAGE: 1100855 3′, AF191339 Homo sapiens anaphase-promotingcomplex subunit 5 (APC5) AA431822 zw79d02.r1 Soares_testis_NHT Homosapiens cDNA clone IMAGE: 782403 NM_003909 Homo sapiens copine III(CPNE3) AA484004 ne73f04.s1 NCI_CGAP_Ew1 Homo sapiens cDNA clone IMAGE:909919 AA535774 nj78f08.s1 NCI_CGAP_Pr10 Homo sapiens cDNA clone IMAGE:998631, mRNA NM_000944.1 Homo sapiens protein phosphatase 3 (formerly2B) AA702811 zi90c11.s1 Soares_fetal_liver_spleen_1NFLS_S1 Homo sapienscDNA X95073 H. sapiens mRNA for translin associated protein X. AA029039zk12b07.s1 Soares_pregnant_uterus_NbHPU Homo sapiens cDNA clone AF032887Homo sapiens forkhead (FKHRL1P1) pseudogene N46609 yy48h08.r1Soares_multiple_sclerosis_2NbHMSP Homo sapiens cDNA U58855 Homo sapienssoluble guanylate cyclase large subunit (GC-S-alpha-1) AA255486zr83d03.s1 Soares_NhHMPu_S1 Homo sapiens cDNA clone IMAGE: 682277AA128153 zl15c06.s1 Soares_pregnant_uterus_NbHPU Homo sapiens cDNA cloneAA016039 ze31c05.s1 Soares retina N2b4HR Homo sapiens cDNA clone R88520ym91e09.s1 Soares adult brain N2b4HB55Y Homo sapiens cDNA clone M26624Human CALLA/NEP gene encoding neutral endopeptidase, exon 20. AA026997ze99e01.r1 Soares_fetal_heart_NbHH19W Homo sapiens cDNA clone W48775zc44b08.r1 Soares_senescent_fibroblasts_NbHSF Homo sapiens cDNA AA074407zm15c08.r1 Stratagene pancreas (#937208) Homo sapiens cDNA clone L13972Homo sapiens beta-galactoside alpha-2,3-sialyltransferase (SIAT4A)D14661 Human mRNA for KIAA0105 gene, complete cds. AA115452 zk89a08.r1Soares_pregnant_uterus_NbHPU Homo sapiens cDNA clone AA495742 zw04b12.r1Soares_NhHMPu_S1 Homo sapiens cDNA clone IMAGE: 768287 5′ R13416yf75h09.r1 Soares infant brain 1NIB Homo sapiens cDNA clone AA046369zk77h07.r1 Soares_pregnant_uterus_NbHPU Homo sapiens cDNA clone T35440EST85129 Human Lung Homo sapiens cDNA 5′ end similar to None, mRNAAI075860 oz25b05.x1 Soares_total_fetus_Nb2HF8_9w Homo sapiens cDNA cloneW56437 zc57g05.r1 Soares_parathyroid_tumor_NbHPA Homo sapiens cDNA cloneAI583880 tt70b02.x1 NCI_CGAP_HSC3 Homo sapiens cDNA clone IMAGE: 22460913′, D85181 Homo sapiens mRNA for fungal sterol-C5-desaturase homolog,complete AF105714 Homo sapiens protein kinase PITSLRE (CDC2L2) gene,exon 3. AA401802 zt60c12.r1 Soares_testis_NHT Homo sapiens cDNA cloneIMAGE: 726742 AB002301 Human mRNA for KIAA0303 gene, partial cds. U75667Human arginase II mRNA, complete cds. AA585183 JTH089 HTCDL1 Homosapiens cDNA 5′/3′, mRNA sequence. AF191771 Homo sapiens CED-6 protein(CED-6) mRNA AA650252 ns93g05.s1 NCI_CGAP_Pr3 Homo sapiens cDNA cloneIMAGE: 1191224, mRNA R64618 yi19b09.r1 Soares placenta Nb2HP Homosapiens cDNA clone N24627 yx89a09.s1 Soares melanocyte 2NbHM Homosapiens cDNA clone AB028951 Homo sapiens mRNA for KIAA1028 proteinN75608 yw37a07.r1 Morton Fetal Cochlea Homo sapiens cDNA clone N53899yy98e03.r1 Soares_multiple_sclerosis_2NbHMSP Homo sapiens cDNA N46696yy50f07.r1 Soares_multiple_sclerosis_2NbHMSP Homo sapiens cDNA AA419522zv03d05.r1 Soares_NhHMPu_S1 Homo sapiens cDNA clone IMAGE: 752553 M61906Human P13-kinase associated p85 mRNA sequence. C16570 C16570 Clontechhuman aorta polyA + mRNA (#6572) Homo sapiens cDNA X63105 H. sapiens tprmRNA. AA315855 EST187656 Colon carcinoma (HCC) cell line II Homo sapienscDNA 5′ L18920 Human MAGE-2 gene exons 1-4, complete cds. M25161 HumanNa, K-ATPase beta subunit (ATP1B) gene AA164865 zq41g07.r1 StratagenehNT neuron (#937233) Homo sapiens cDNA clone N40094 yx98g07.r1 Soaresmelanocyte 2NbHM Homo sapiens cDNA clone N98940 yy71a07.r1Soares_multiple_sclerosis_2NbHMSP Homo sapiens cDNA AF049907 Homosapiens zinc finger transcription factor (ZNF-X) mRNA, M78806 EST00954Hippocampus, Stratagene (cat. #936205) Homo sapiens cDNA AA040819zk47b03.r1 Soares_pregnant_uterus_NbHPU Homo sapiens cDNA clone C15445C15445 Clontech human aorta polyA + mRNA (#6572) Homo sapiens cDNAAB018309 Homo sapiens mRNA for KIAA0766 protein, complete cds. AJ011497Homo sapiens mRNA for Claudin-7. X00949 Human mRNA for prepro-relaxinH1. AA418633 zv93d09.r1 Soares_NhHMPu_S1 Homo sapiens cDNA clone IMAGE:767345 5′ AI146806 qb83h04.x1 Soares_fetal_heart_NbHH19W Homo sapienscDNA clone X82942 H. sapiens satellite 3 DNA. AA456383 aa14f03.r1Soares_NhHMPu_S1 Homo sapiens cDNA clone IMAGE: 813245 AA019341ze57e04.s1 Soares retina N2b4HR Homo sapiens cDNA clone AB027466 Homosapiens SPON2 mRNA for spondin 2 AF038170 Homo sapiens clone 23817 mRNAsequence. NM_000240 Homo sapiens monoamine oxidase A (MAOA) N34126yx76c01.r1 Soares melanocyte 2NbHM Homo sapiens cDNA clone N41339yw68g06.r1 Soares_placenta_8to9weeks_2NbHP8to9W Homo sapiens cDNA R34783yh87b05.r1 Soares placenta Nb2HP Homo sapiens cDNA clone N75858yw32a03.r1 Morton Fetal Cochlea Homo sapiens cDNA clone AA633887ac32h04.s1 Stratagene hNT neuron (#937233) Homo sapiens cDNA cloneN53723 yz06d03.r1 Soares_multiple_sclerosis_2NbHMSP Homo sapiens cDNAAI187365 qf29b12.x1 Soares_testis_NHT Homo sapiens cDNA clone IMAGE:1751423Genes in bold type are known prostate-specific genes.

TABLE 5 Genes/ESTs as Defined by Publications: Including AndrogenSignaling, Prostate Specificity, Prostate Cancer Association, andNuclear Receptors/Regulators with Potential Interaction with AndrogenReceptor Cluster ID Gene Name Description References Hs.81988 DOC-2deliion of ovarian Up-regulated by Androgen Ablation Endocrinology,carcinoma 2 139,3542,98 Hs.155389 RAR a Up-regulated by AndrogenAblation endocrinology, 138,553,97 Hs.12601 AS3 DNA binding proteinUp-regulated by Androgen Ablation J Steroid Biochem Mol Biol 68,41,99Hs.181426 EST Up-regulated by Androgen Ablation Hs.2391 apical proteinUp-regulated by Androgen Ablation Hs.109530 KGF/FGF7 keratinocyte growthfactor Up-regulated by Androgen BBRC 220,858,96, Can Res, 54,5474,94Hs.1104 TGF beta 1 Up-regulated by Androgen Endocrinology, 137,99,96,Endocrinology, 139,378,98 Hs.75525 Calreticulin CalreticulinUp-regulated by Androgen Can Res 59,1896,99 Hs.78888 DBI/ACBPDiazepam-binding Up-regulated by Androgen JBC, 237,19938,98inhibitor/acyl-CoA binding Protein Hs.41569 Phosphatidic acidUp-regulated by Androgen JBC, 273,4660,98 phosphatase type 2a isozymeHs.83190 Fatty acid synthase Up-regulated by Androgen Can Res,57,1086,97 Hs.99915 Androgen Receptor Up-regulated by Androgen Steroids9,531,96 Hs.2387 prostate-restricted Up-regulated by Androgen Biochem J315,901,96 transglutaminase Hs.78996 PCNA proliferating cellUp-regulated by Androgen Can Res 56,1539,96 nuclear antigen Hs.74456GAPDH Up-regulated by Androgen Can Res 55,4234,95 Hs.82004 E cadherinUp-regulated by Androgen BBRC, 212,624,95 Hs.57710 AIGF Androgen-inducedUp-regulated by Androgen FEBS lett 363,226,95 growth factor Hs.118618MIC2 humanpseudoautosom Up-regulated by Androgen Mol Carcinog, al gene?23,13,98 Hs.18420 Talin cytoskeletal protein Up-regulated by AndrogenFEBS lett 434,66,98 Hs.54502 clathrin heavy chain Up-regulated byAndrogen Endocrinology, 139,2111,98 Hs.73919 clathrin light chain bUp-regulated by Androgen Endocrinology, 139,2111,98 Hs.76506 L-plastinESTs, Moderately Up-regulated by Androgen Am J Pathol, 150, similar toL- 2009,97 PLASTIN [H. sapiens] Hs.82173 EGR alpha TGFB inducible earlyUp-regulated by Androgen Mol Endocrinol, growth response 9,1610,95 NDFGF10 Up-regulated by Androgen JBC, 274,12827,99 Hs.107169 IGFBP5Up-regulated by Androgen Endocrinology, 140,2372,99 Hs.179665 p21Up-regulated by Androgen Mol Endocrinol, 13,376,99 Hs.51117 BMP-7Up-regulated by Androgen Prostate, 37,236,98 Hs.73793 VEGF vascularendothelial Up-regulated by Androgen Endocrinol, 139,4672,98, growthfactor BBRC, 251,287,98 Hs.166 SREBPs sterol regulatory Up-regulated byAndrogen J Steroid Biochem Mol element binding Biol, 65,191,98transcription factor1 Hs.116577 PDF prostate Up-regulated by AndrogenJBC, 273,13760,98 differentiation factor Hs.1905 prolactin ProlactinUp-regulated by Androgen FEBS J, 11,1297,97 Hs.19192 CDK2 Up-regulatedby Androgen Can Res, 57,4511,97 Hs.95577 CDK4 cyclin-dependentUp-regulated by Androgen Can Res, 57,4511,97 kinase 4 Hs.183596 UGT2B17uridine Up-regulated by Androgen Endocrinology, diphosphoglucronosyl138,2998,97 transferase Hs.150207 UGT2B15 UDP- Up-regulated by AndrogenCan Res 57,4075,97 glucronosyltransferase 2B15 ND prostate bindingprotein Up-regulated by Androgen PNAS, 94,12999,97 C2A (RAT) ND Probasin(RAT) Up-regulated by Androgen PNAS, 94,12999,97 Hs.7719 prostatein C3(RAT) Up-regulated by Androgen PNAS, 94,12999,97 ND Cystatin relatedprotein 1 Up-regulated by Androgen PNAS, 94,12999,97 (RAT) ND Cystatinrelated protein 2 Up-regulated by Androgen PNAS, 94,12999,97 (RAT)Hs.394 Adrenomedulin (RAT) Up-regulated by Androgen PNAS, 94,12999,97Hs.77393 farnesyl diphosphate Up-regulated by Androgen PNAS, 94,12999,97synthase (farnesyl pyrophosphate synthetase,dimethylallyltranstransferase) Hs.153468 LDL receptor (Rat) Up-regulatedby Androgen PNAS, 94,12999,97 N.D. Hysto-blood group A Up-regulated byAndrogen PNAS, 94,12999,97 transferase (RAT) Hs.196604 Sex limitedprotein Up-regulated by Androgen PNAS, 94,12999,97 (RAT) slp NDprostatic spermine Up-regulated by Androgen Mol Cell Endocrinol, bindingprotein(RAT) 108, R1, 95 Hs.76353 Protein C Inhibitor Up-regulated byAndrogen FEBS lett, 492,263,98 Hs.203602 enolase alpha Up-regulated byAndrogen Can Res, 58,5718,98 Hs.169476 tubulin alpha Up-regulated byAndrogen Can Res, 58,5718,98 Hs.184572 Cdk1 Up-regulated by Androgen CanRes, 58,5718,98 Hs.107528 EST EST similar to Up-regulated by Androgenandrogen-regulated protein FAR-17 Hs.28309 UDP-glucose Up-regulated byAndrogen Endocrinology, dehydrogenase 140,10,4486,(99) Hs.194270secretory component Up-regulated by Androgen Mol endocrinol, gene13,9,1558,(99) Hs.76136 Thioredoxin Up-regulated by Androgen J steroidBiochem Mol Biol, 68, 5-6, 203, (99) Hs.3561 p27 Kip1 cyclin-dependentUp-regulated by Androgen Mol kinase inhibitor 1B Endocrinol, 12,941,98(p27, Kip1) Hs.1867 progastricsin Up-regulated by Androgen J.B.C.271,15175,(99) (pepsinogen C) Hs.97411 hamster Androgen- Up-regulated byAndrogen Genebank dependent Expressed Protein like protein geneHs.155140 Protein kinase CK2 casein kinase 2, alpha Translocated byAndrogen Can Res 59,1146,99 1 polypeptide IMAGE: 953262 DD3 ProstateSpecific Eur Urol, 35,408,99 Hs.218366 Prostase Prostate Specific PNAS,96,3114,99 Hs.20166 PSCA prostate stem cell Prostate Specific PNAS,95,1735,98 antigen Hs.171995 PSA kallikrein 3, (prostate ProstateSpecific PNAS, 95,300,98, specific antigen) DNA Cell Biol, 16,627,97Hs.183752 PSSPP prostate-secreted Prostate Specific PNAS, 95,300,98seminal plasma protein, nc50a10, microsemnoprotein beta, PSP94 Hs.1852PAP prostatic acid Prostate Specific PNAS, 95,300,98 phosphataseHs.52871 SYT Prostate Specific PNAS, 95,300,98 Hs.158309 Homeobox HOXD13 Prostate Specific PNAS, 95,300,98 Hs.1968 Semenogelin 1 ProstateSpecific PNAS, 95,300,98 Hs.76240 Adenylate kinase adenylate kinase 1Prostate Specific PNAS, 95,300,98 isoenzyme1 Hs.184376 SNAP23 ProstateSpecific PNAS, 95,300,98 Hs.82186 ERBB-3 receptor Prostate SpecificPNAS, 95,300,98 protein-tyrosin kinase Hs.180016 Semenogelin 2 ProstateSpecific Hs.1915 PSMA folate hydrolase Prostate Specific(prostate-specific membrane antigen) 1 Hs.181350 KLK2 Prostate SpecificHs.73189 NKX3.1 Prostate Specific HPARJ1 Prostate Specific IMAGE: 565779Hs.76053 p68 RNA helicase Potential interaction with AR MCB,19,5363,(99) Hs.111323 ARIP3 Potential interaction with AR JBC,274,3700,99 Hs.25511 ARA54 Potential interaction with AR JBC274,8319,99Hs.28719 ARA55 Potential interaction with AR JBC, 274,8570,99 HS. 999908ARA70 Potential interaction with AR PNAS, 93,5517,96 Hs.29131 TIF2transcriptional Potential interaction with AR EMBO, 15,3667,96,intermediary factor 2 EMBO, 17,507,98 Hs.66394 SNURF ring finger protein4 Potential interaction with AR MCB, 18,5128,98 Hs.75770 RBretinoblastoma 1 Potential interaction with AR (including osteosarcoma)Hs.74002 SRC-1 steroid receptor Potential interaction with ARcoactivator 1 Hs.155017 RIP140 nuclear receptor Potential interactionwith AR EMBO, 14,3741,95, interacting protein 1 Mol Endocrinol,12,864,98 Hs.23598 CBP CREB binding Potential interaction with ARprotein (Rubinstein- Taybi syndrome) Hs.25272 p300 E1A binding proteinPotential interaction with AR p300 Hs.78465 c-JUN Potential interactionwith AR Hs.199041 ACTR AIB1, mouse Potential interaction with AR M.C.B,17,2735,97, GRIP1, pCIP PNAS, 93,4948,96 Hs.6364 TIP60 Human tatinteractive Potential interaction with AR JBC, 274,17599,99 proteinmRNA, complete cds Hs.32587 SRA Potential interaction with AR Cell,97,17,99 Hs.155302 PCAF Potential interaction with AR Hs.10842 ARA24Potential interaction with AR Hs.41714 BAG-IL Potential interaction withAR JBC, 237,11660,98 Hs.82646 dnaJ, HSP40 DNAJ PROTEIN Potentialinteraction with AR HOMOLOG 1 Hs.43697 ERM ets variant gene 5 Potentialinteraction with AR JBC, 271,23907,96 (ets-related molecule) Hs.75772 GRPotential interaction with AR JBC, 272,14087,97 Hs.77152 MCM7 Potentialinteraction with AR ND NJ Potential interaction with AR ND RAF Potentialinteraction with AR JBC, 269,20622,94 ND TFIIF Potential interactionwith AR PNAS, 94,8485,97 Hs.90093 hsp70 Potential interaction with ARHs.206650 hsp90 Potential interaction with AR Hs.848 hsp56(FKBP52,Potential interaction with AR FKBP59, HBI)) Hs.143482Cyp40(cyclophilin40) Potential interaction with AR p23 Potentialinteraction with AR Hs.84285 ubiquitin-conjugating Potential Interactionwith AR J.B.C. 274,19441(99) enzyme Hs.182237 POU domain, class 2,Potential interaction with AR transcr Hs.1101 POU domain, class 2,Potential interaction with AR transcr Hs.2815 POU domain, class 6,Potential interaction with AR transcr IMAGE: 1419981 Potentialinteraction with AR Hs.227639 ARA160 Potential interaction with AR JBC,274,22373(99) Hs.83623 CAR-beta Xist locus Nuclear receptor gene familyHs.2905 PR Nuclear receptor gene family Hs.1790 MR mineralocorticoidNuclear receptor gene family receptor (aldosterone receptor) Hs.1657 ERalpha Nuclear receptor gene family Hs.103504 ER beta Nuclear receptorgene family Hs.110849 ERR1 Nuclear receptor gene family Hs.194667 ERR2Nuclear receptor gene family Hs.724 TR a thyroid hormone Nuclearreceptor gene family receptor, alpha (avian erythroblastic leukemiaviral (v-erb- a) oncogene homolog) Hs.121503 TR b Nuclear receptor genefamily Hs.171495 RAR b retinoic acid receptor, Nuclear receptor genefamily beta Hs.1497 RAR g retinoic acid receptor, Nuclear receptor genefamily gamma Hs.998 PPAR a Nuclear receptor gene family Hs.106415 PPAR bHuman peroxisome Nuclear receptor gene family proliferator activatedreceptor mRNA, complete cds Hs.100724 PPAR g peroxisome Nuclear receptorgene family proliferative activated receptor, gamma Hs.100221 LXR bNuclear receptor gene family Hs.81336 LXR a liver X receptor, Nuclearreceptor gene family alpha Hs.171683 FXR farnesoid X-activated Nuclearreceptor gene family receptor Hs.2062 VDR vitamin D (1,25- Nuclearreceptor gene family dihydroxyvitamin D3) receptor Hs.118138 PXR Nuclearreceptor gene family ND SXR Nuclear receptor gene family ND BXR Nuclearreceptor gene family ND CAR b? CAR a Nuclear receptor gene familyHs.196601 RXRA Nuclear receptor gene family Hs.79372 RXRB Human retinoidX Nuclear receptor gene family receptor beta (RXR- beta) mRNA, completecds Hs.194730?TR1? EAR1 Nuclear receptor gene family Hs.204704 EAR1 betaNuclear receptor gene family E75 Nuclear receptor gene family Hs.2156ROR alpha Nuclear receptor gene family Hs.198481 ROR beta Nuclearreceptor gene family Hs.133314 ROR gammma Nuclear receptor gene familyHs.100221 NER1 Nuclear receptor gene family Hs.54424 HNF4A Nuclearreceptor gene family Hs.202659 HNF4G Nuclear receptor gene familyHs.108301 TR2 Nuclear receptor gene family Hs.520 TR4 Nuclear receptorgene family Hs.144630 COUP-TF1 Nuclear receptor gene family Hs.1255COUP-TF2 Nuclear receptor gene family Hs.155286 EAR2 Nuclear receptorgene family Hs.1119 TR3 hormone receptor Nuclear receptor gene family(growth factor- inducible nuclear protein N10) Hs.82120 NURR1 IMMEDIATE-Nuclear receptor gene family EARLY RESPONSE PROTEIN NOT Hs.97196 SF1Nuclear receptor gene family Hs.183123 FTF fetoprotein-alpha 1 Nuclearreceptor gene family (AFP) transcription factor Hs.46433 DAX1 Nuclearreceptor gene family Hs.11930 SHP Homo sapiens nuclear Nuclear receptorgene family hormone receptor (shp) gene, 3′ end of cds Hs.83623,CAR-beta Nuclear receptor gene family IMAGE 1761923, or 1868028, or1563505, or 1654096 Hs.199078 Sin3 Nuclear receptor co-repressor complexNature, 387,43,97, Nature, 387,49,97 Hs.120980 SMRT Nuclear receptorco-repressor complex Nature, 377,454,95 Hs.144904 N-CoR Nuclear receptorco-repressor complex Nature, 377,297,95 Hs.188055 highly homologue geneNuclear receptor co-repressor complex to N-CoR in prostate and testisHs.180686 E6-AP Angelman syndrome Nuclear receptor co-activator complexMCB, 19,1182,99 associated protein Hs.199211?Hs. hBRM ESTs, Highlysimilar Nuclear receptor co-activator complex 198296? to HOMEOTIC GENEREGULATOR [Drosophila melanogaster] Hs.78202 hBRG1 Nuclear receptorco-activator complex Hs.11861 TRAP240 DRIP250, ARCp250 Nuclear receptorco-activator complex Mol Cell, 3,361,99 Hs.85313 TRAP230 ARCp240,DRIP240 Nuclear receptor co-activator complex Mol Cell, 3,361,99Hs.15589 TRAP220 RB18A, PBP, Nuclear receptor co-activator complexCRSP200, TRIP2, ARCp205, DRIP205 Hs.21586 TRAP170 RGR, CRSP150, Nuclearreceptor co-activator complex DRIP150, ARCp150 chromosomeX Hs.108319TRAP150 ESTs Nuclear receptor co-activator complex Mol Cell, 3,361,99Hs.193017 CRSP133 ARCp130, DRIP130 Nuclear receptor co-activator complexNature, 397,6718,99 Hs.23106 TRAP100 ARCp100, DRIP100, Nuclear receptorco-activator complex ND DRIP97 TRAP97 Nuclear receptor co-activatorcomplex Hs.24441 TRAP95 ESTs Nuclear receptor co-activator complex MolCell, 3,361,99 ND TRAP93 Nuclear receptor co-activator complex Hs.31659DRIP92 ARCp92? Nuclear receptor co-activator complex Hs.22630 TRAP80ARCp77, Nuclear receptor co-activator complex Mol Cell, 3,361,99 CRSP77,DRIP80(77)? Hs.204045 ARCp70 CRSP70, DRIP70 Nuclear receptorco-activator complex ND ARCp42 Nuclear receptor co-activator complex NDARCp36 Nuclear receptor co-activator complex Hs.184947 MED6 ARCp33Nuclear receptor co-activator complex Mol Cell, 3,97,99 Hs.7558 MED7CRSP33, ARCp34, Nuclear receptor co-activator complex Nature,397,6718,99 DRIP36 ND ARCp32 Nuclear receptor co-activator complex NDSRB10 Nuclear receptor co-activator complex ND SRB11 Nuclear receptorco-activator complex ND MED10 NUT2 Nuclear receptor co-activator complexHs.27289 SOH1 (yeast?) Nuclear receptor co-activator complex Mol Cell,3,97,99 ND p26 Nuclear receptor co-activator complex ND p28 Nuclearreceptor co-activator complex ND p36 Nuclear receptor co-activatorcomplex ND p37 Nuclear receptor co-activator complex ND but 2 TRFP humanhomologue of Nuclear receptor co-activator complex IMAGE clonesDrosophila TRF proximal protein ND VDR interacting subunit 180 kDa, HATNuclear receptor co-activator complex Genes Dev, 12,1787,98 activityHs.143696, or Coactivator associated Nuclear receptor co-activatorcomplex Science, 284,2174,99 IMAGE: 23716 methyltransferase 1 96?Hs.79387 SUG1 TRIP1 Nuclear receptor co-activator complex EMBO,15,110,96 ND TRUP Nuclear receptor co-activator complex PNAS, 92,9525,95Hs.28166 CRSP34 Nuclear receptor co-activator complex Nature,397,6718,99 Hs.63667 transcriptional adaptor 3 Nuclear receptorco-activator complex (A Hs.196725 ESTs, Highly similar to Nuclearreceptor co-activator complex P300 Hs.131846 PCAF associated factorNuclear receptor co-activator complex 65 al Hs.155635 ESTs, ModeratelyNuclear receptor co-activator complex similar to PCAF associated factor65 beta Hs.26782 PCAF associated factor Nuclear receptor co-activatorcomplex 65 beta Hs.118910 tumor suscitibility Modifying AR functionCancer 15,86,689, protein 101 (99) Hs.82932 Cyclin D1 cyclin D1 (PRADI:Modifying AR function Can Res, 59,2297,99 parathyroid adenomatosis 1)Hs.173664 HER2/Neu v-erb-b2 avian Modifying AR function PNAS, 9,5458,99erythroblastic leukemia viral oncogene homolog 2 Hs.77271 PKA proteinkinase, Modifying AR function JBC 274,7777,99 cAMP-dependent, catalytic,alpha Hs.85112 IGF1 insulin-like growth Modifying AR function Can Res,54,5474,94 factor 1 (somatomedin C) Hs.2230 EGF Modifying AR functionCan Res, 54,5474,94 Hs.129841 MEKK1 MAPKKK1 Modifying AR function MolCell Biol, 19,5143,99 Hs.83173 Cyclin D3 Modifying AR function Can Res,59,2297,99 Hs.75963 IGF2 Modifying AR function Hs.89832 InsulinModifying AR function Hs.115352 GH Modifying AR function Hs.1989 5 alphareductase type2 Involved in Androgen metabolism Hs.76205 CytochromeP450, Involved in Androgen metabolism subfamily XIA Hs.1363 CytochromeP450, Involved in Androgen metabolism subfamily XVII, (steroid17-alpha-hydroxylase), Hs.477 Hydroxysteroid (17- Involved in Androgenmetabolism beta) dehydrogenase 3 Hs.75441 Hydroxysteroid (17- Involvedin Androgen metabolism beta) dehydrogenase 4 Hs.38586Hydroxy-delta-5-steroid Involved in Androgen metabolism dehydrogenase, 3beta- and steroid delta- isomerase 1 Hs.46319 Sex hormone-bindingInvolved in Androgen metabolism globulin Hs.552 SRD5A1 Involved inAndrogen metabolism Hs.50964 C-CAM epithelial cell Down-regulated byAndrogen Oncogene, 18,3252,99 adhesion molecule Hs.7833 hSP56 seleniumbinding Down-regulated by Androgen Can Res, 58,3150,98 protein Hs.77432EGFR epidermal growth Down-regulated by Androgen Endocrinology, factorreceptor 139,1369,98 Hs.1174 p16 Down-regulated by Androgen Can Res,57,4511,97 Hs.55279 maspin Down-regulated by Androgen PNAS, 94,5673,97Hs.75789 TDD5 (mouse) Human mRNA for Down-regulated by Androgen PNAS,94,4988,97 RTP, complete cds Hs.75106 TRPM-2 clusterin ( Down-regulatedby Androgen testosterone-repressed prostate message 2, apolipoprotein J)Hs.25640 rat ventral prostate gene 1 claudin3 Down-regulated by AndrogenPNAS, 94,12999,97 ND glutathione S-transferase Down-regulated byAndrogen PNAS, 94,12999,97 Hs.25647 c-fos v-fos FBJ murineDown-regulated by Androgen PNAS, 94,12999,97 osteosarcoma viral oncogenehomolog N.D. matrix carboxyglutamic Down-regulated by Androgen PNAS,94,12999,97 acid protein (RAT) Hs.2962 S100P calcium bindingDown-regulated by Androgen Prostate 29,350,96 prottein Hs.75212ornithine decarboxilase ornithine Down-regulated by Androgen J Androl,19,127,98 decarboxylase 1 Hs.84359 Androge withdrawal Down-regulated byAndrogen apoptosis RVP1 Hs.79070 c-myc v-myc avian Down-regulated byAndrogen myelocytomatosis viral oncogene homolog Hs.139033 partiallyexpressed gene 3 Down-regulated by Androgen Mol Cell Endocrinol155,69,(99) Hs.20318 PLU-1 Associated with Prostate Cancer JBC,274,15633.99 Hs.18910 POV1(PB39) unique Associated with Prostate CancerGenomics, 51,282,98 Hs.119333 caveolin Associated with Prostate CancerClin Can Res, 4, 1873,98 ND, but 1 EST R00540(2.6 kbp) = 1M Associatedwith Prostate Cancer Urology, 50,302,97 IMAGE AGE: 123822 CLONEHs.184906 PTI-1 prostate tumor Associated with Prostate Cancer Can Res,57,18,97, inducing gene, PNAS, 92,6778,95 trancated and mutated humanelongation factor 1 alpha Hs.74649 cytochrome c oxidase Associated withProstate Cancer Can Res, 56,3634,96 subunit VI c Hs.4082 PCTA-1 prostatecarcinoma Associated with Prostate Cancer PNAS, 92,7252,96 tumorantigen, galectin family ND pp32r1 Associated with Prostate CancerNature Medicine, 5,275,99 ND pp32r2 Associated with Prostate CancerNature Medicine, 5,275,99 Hs.184945 GBX2 Associated with Prostate CancerThe prostate journal, 1,61,99 Hs.8867 Cyr61 inmmediate early Associatedwith Prostate Cancer Prostate, 36,85,98 protein Hs.77899 epithelialtropomyosin actin binding protein Associated with Prostate Cancer CanRes, 56,3634,96 Hs.76689 pp32 Associated with Prostate Cancer NatureMedicine, 5,275,99 Hs.10712 PTEN Associated with Prostate CancerHs.194110 KAI1 Associated with Prostate Cancer Hs.37003 H-ras Associatedwith Prostate Cancer Hs.184050 K-ras Associated with Prostate CancerHs.69855 N-ras neuroblastoma RAS Associated with Prostate Cancer viral(v-ras) oncogene homolog Hs.220 TGFbeta receptor1 Associated withProstate Cancer Hs.77326 IGFBP3 insulin-like growth Associated withProstate Cancer factor binding protein 3 Hs.79241 bcl-2 Associated withProstate Cancer Hs.159428 Bax Associated with Prostate Cancer Hs.206511bcl-x Associated with Prostate Cancer Hs.86386 mcl-1 myeloid cellleukemia Associated with Prostate Cancer sequence 1 (BCL2- related)Hs.1846 p53 tumor protein p53 Associated with Prostate Cancer(Li-Fraumeni syndrome) Hs.38481 CDK6 cyclin-dependent Associated withProstate Cancer kinase 6 Hs.118630 Mxi.1 Associated with Prostate CancerHs.184794 GAGE7 Associated with Prostate Cancer Hs.118162 fibronectinAssociated with Prostate Cancer Am J Pathol 154,1335,99 Hs.128231 PAGE-1Associated with Prostate Cancer JBC, 237,17618,98 Hs.75875 UEV1ubiquitin-conjugating Associated with Prostate Cancer Am J Pathol enzymeE2 variant 1 154,1335,99 Hs.75663 PM5 Human mRNA for Associated withProstate Cancer Am J Pathol pM5 protein 154,1335,99 Hs.180842 BBC1breast basic Associated with Prostate Cancer Am J Pathol conserved gene154,1335,99 Hs.198024 JC19 Associated with Prostate Cancer Can Res57,4075,97 N.D. GC79 novel gene Associated with Prostate Cancer Can Res57,4075,97 Hs.77054 B cell translocation gene 1 Associated with ProstateCancer Can Res 57,4075,97 Hs.78122 Regulatory factor X- Associated withProstate Cancer associated ankyrin- containing protein Hs.3337transmembranc 4 Associated with Prostate Cancer superfamily member1Hs.76698 TL5 Associated with Prostate Cancer Genebank Hs.3776 TL7Associated with Prostate Cancer Genebank Hs.170311 TL35 Associated withProstate Cancer Genebank Hs.184914 Human mRNA for T1- Associated withProstate Cancer 227H Hs.62954 ferritin, heavy Associated with ProstateCancer polypeptide Hs.71119 N33 Associated with Prostate CancerGenomics, 35,45(96)

TABLE 6 Genes/ESTs as defined by publications: Differentially expresedgenes in prostate cancer from CGAP database (NIH) Cluster.ID Gene nameHs.179809 EST Hs.193841 EST Hs.99949 prolactin-induced protein Hs.101307EST Hs.111256 arachidonate 15-lipoxygenase Hs.185831 EST Hs.115173 ESTHs.193988 EST Hs.159335 EST Hs.191495 EST Hs.187694 EST Hs.191848 ESTHs.193835 EST Hs.191851 EST Hs.178512 EST Hs.222886 EST Hs.210752 ESTHs.222737 EST Hs.105775 EST Hs.115129 EST Hs.115671 EST Hs.116506 ESTHs.178507 EST Hs.187619 EST Hs.200527 EST Hs.179736 EST Hs.140362 ESTHs.209643 EST Hs.695559 EST Hs.92323 MAT8 Hs.178391 BTK Hs.55999 ESTHs.171185 Desmin Hs.54431 SGP28 Hs.182624 EST Hs.112259 T cell receptorgammma Hs.76437 EST Hs.104215 EST Hs.75950 MLCK Hs.154103 LIM Hs.9542JM27 Hs.153179 FABP5 Hs.195850 EST Hs.105807 EST Hs.115089 EST Hs.116467EST Hs.222883 EST

TABLE 7 Androgen regulated Genes Defined by CPDR Genes/ESTs Derived fromCPDR-Genome Systems ARG Database Cluster Gene Name Description Hs.152204TMPRSS2 Up-regulated by Androgen Hs.123107 KLK1 Up-regulated by AndrogenHs.173334 elongation factor ell2 Up-regulated by Androgen Hs.151602epithelial V-like antigen Up-regulated by Androgen Hs.173231 IGFRIUp-regulated by Androgen Hs.75746 aldehyde dehydrogenase 6 Up-regulatedby Androgen Hs.97708 EST prostate and testis Up-regulated by AndrogenHs.94376 proprotein convertase subtilisin/kexin type 5 Up-regulated byAndrogen AF017635 Homo sapiens Ste-20 related kinase SPAK mRNA, completecds {Incyte PD: Up-regulated by Androgen 60737} Hs.2798 leukemiainhibitory factor receptor Up-regulated by Androgen Hs.572 orosomucoid 1Up-regulated by Androgen Hs.35804 KIAA0032 gene product Up-regulated byAndrogen Hs.114924 solute carrier family 16 (monocarboxylic acidtransporters), member 6 Up-regulated by Androgen Hs.37096 zinc fingerprotein 145 (Kruppel-like, expressed in promyelocytic leukemia)Up-regulated by Androgen R07295 sterol O-acyltransferase (acyl-CoenzymeA: cholesterol acyltransferase) 1 Up-regulated by Androgen {Incyte PD:2961248} Hs.11899 3-hydroxy-3-methylglutaryl-Coenzyme A reductaseUp-regulated by Androgen Hs.216958 Human mRNA for KIAA0194 gene, partialcds Up-regulated by Androgen Hs.76901 for protein disulfideisomerase-related Up-regulated by Androgen Hs.180628 dynamin-likeprotein Up-regulated by Androgen Hs.81328 nuclear factor of kappa lightpolypeptide gene enhancer in B-cells inhibitor, Up-regulated by Androgenalpha Hs.159358 acetyl-Coenzyme A carboxylase alpha Up-regulated byAndrogen N24233 IMAGE: 262457 Up-regulated by Androgen Hs.188429 ESTUp-regulated by Androgen Hs.77508 glutamate dehydrogenase 1 Up-regulatedby Androgen Hs.12017 Homo sapiens KIAA0439 mRNA Up-regulated by AndrogenHs.10494 EST Up-regulated by Androgen Hs.20843 EST Up-regulated byAndrogen Hs.153138 origin recognition complex, subunit 5 (yeasthomolog)-like Up-regulated by Androgen Hs.79136 Human breast cancer,estrogen regulated LIV-1 protein (LIV-1) mRNA, partial Up-regulated byAndrogen cds Hs.35750 anthracycline resistance-associated Up-regulatedby Androgen Hs.56729 lymphocyte-specific protein 1 Up-regulated byAndrogen Hs.17631 EST Up-regulated by Androgen Hs.46348 bradykininreceptor B1 Up-regulated by Androgen Hs.172851 arginase, type IIUp-regulated by Androgen Hs.66744 twist (Drosophila) homologUp-regulated by Androgen Hs.185973 membrane fatty acid (lipid)desaturase Up-regulated by Androgen Hs.26 ferrochelatase(protoporphyria) Up-regulated by Androgen Hs.169341 ESTs, Weakly similarto phosphatidic acid phosphohydrolase type-2c Up-regulated by Androgen[H. sapiens] Hs.119007 S-phase response (cyclin-related) Up-regulated byAndrogen Hs.76285 H. sapiens gene from PAC 295C6, similar to rat PO44Up-regulated by Androgen Hs.167531 Homo sapiens mRNA full length insertcDNA clone EUROIMAGE 195423 Up-regulated by Androgen Hs.9817arg/Abl-interacting protein ArgBP2 Up-regulated by Androgen Hs.28241 ESTDown-regulated by Androgen Hs.25925 Homo sapiens clone 23860 mRNADown-regulated by Androgen Hs.10319 UDP glycosyltransferase 2 family,polypeptide B7 Down-regulated by Androgen Hs.155995 Homo sapiens mRNAfor KIAA0643 protein, partial cds Down-regulated by Androgen Hs.23552EST Down-regulated by Androgen Hs.41693 DnaJ-like heat shock protein 40Down-regulated by Androgen Hs.90800 matrix metalloproteinase 16(membrane-inserted) Down-regulated by Androgen Hs.2996sucrase-isomaltase Down-regulated by Androgen Hs.166019 regulatoryfactor X, 3 (influences HLA class II expression) Down-regulated byAndrogen Hs.27695 midline 1 (Opitz/BBB syndrome) Down-regulated byAndrogen Hs.183738 chondrocyte-derived ezrin-like protein Down-regulatedby Androgen Hs.75761 SFRS protein kinase 1 Down-regulated by AndrogenHs.197298 NS1-binding protein Down-regulated by Androgen Hs.149436kinesin family member 5B Down-regulated by Androgen Hs.81875 growthfactor receptor-bound protein 10 Down-regulated by Androgen Hs.75844ESTs, Weakly similar to (defline not available 5257244) [H. sapiens]Down-regulated by Androgen Hs.30464 cyclin E2 Down-regulated by AndrogenHs.198443 inositol 1,4,5-triphosphate receptor, type 1 Down-regulated byAndrogen Hs.177959 a disintegrin and metalloproteinase domain 2(fertilin beta) Down-regulated by Androgen Hs.44197 Homo sapiens mRNA;cDNA DKFZp564D0462 (from clone Down-regulated by Androgen DKFZp564D0462)Hs.150423 cyclin-dependent kinase 9 (CDC2-related kinase) Down-regulatedby Androgen Hs.78776 Human putative transmembrane protein (nma) mRNA,complete cds Down-regulated by Androgen Hs.25740 ESTs, Weakly similar to!!!! ALU SUBFAMILY SQ WARNING ENTRY !!!! Down-regulated by Androgen [H.sapiens] Hs.131041 EST Down-regulated by Androgen Hs.19222 ecotropicviral integration site 1 Down-regulated by Androgen Hs.9879 ESTDown-regulated by Androgen Hs.118722 fucosyltransferase 8 (alpha (1,6)fucosyltransferase) Down-regulated by Androgen Hs.47584 potassiumvoltage-gated channel, delayed-rectifier, subfamily S, member 3Down-regulated by Androgen Hs.115945 mannosidase, beta A, lysosomalDown-regulated by Androgen Hs.171740 ESTs, Weakly similar to Zic2protein [M. musculus] Down-regulated by Androgen Hs.32970 signalinglymphocytic activation molecule Down-regulated by Androgen Hs.196349 ESTDown-regulated by Androgen Hs.182982 Homo sapiens mRNA for KIAA0855protein, partial cds Down-regulated by Androgen Hs.72918 small induciblecytokine A1 (I-309, homologous to mouse Tca-3) Down-regulated byAndrogen Hs.84232 transcobalamin II; macrocytic anemia Down-regulated byAndrogen Hs.10086 EST Down-regulated by Androgen Hs.1327Butyrylcholinesterase Down-regulated by Androgen Hs.166684serine/threonine kinase 3 (Ste20, yeast homolog) Down-regulated byAndrogen AA558631 EST Down-regulated by Androgen Hs.150403 dopadecarboxylase (aromatic L-amino acid decarboxylase) Down-regulated byAndrogen Hs.177548 postmeiotic segregation increased (S. cerevisiae) 2Down-regulated by Androgen

TABLE 8 Other Genes Associated with Cancers Cluster Gene nameDescription Hs.146355 c-Abl v-abl Abelson murine leukemia viral oncogenehomolog 1 Hs.96055 E2F1 Hs.170027 MDM2 Hs.1608 RPA replication proteinA3 (14 kD) Hs.99987 XPD ERCC2 Hs.77929 XPB ERCC3 Hs.1100 TBP TATA boxbinding protein Hs.60679 TAFII31 TATA box binding protein(TBP)-associated factor, RNA polymerase II, G, 32 kD Hs.78865 TAFII70Human TBP-associated factor TAFII80 mRNA, complete cds Hs.178112 DP1deleted in poliposis Hs.119537 p62 Hs.48576 CSB excision repaircross-complementing rodent repair deficiency, complementation group 5Hs.73722 Ref-1 Hs.194143 BRCA1 breast cancer 1, early onset Hs.184760CBF Hs.1145 WT-1 Wilms tumor 1 Hs.2021 Sp1 Hs.144477 CK I Hs.155627DNA-PK Hs.170263 p53BP1 Human clone 53BP1 p53-binding protein mRNA,partial cds Hs.44585 p53BP2 tumor protein p53-binding protein, 2 Hs.6241p85 alpha PI3 kinase Hs.23707 p85 beta PI3 kinase Hs.194382 ATMHs.184948 BIN1 Hs.137569 p51B p63 Hs.1334 bmyb v-myb avianmyeloblastosis viral oncogene homolog Hs.81942 DNA polymerase polymerase(DNA directed), alpha alpha Hs.180952 Beta actin Hs.93913 IL-6interleukin 6 (interferon, beta 2) Hs.190724 MAP4 microtubule-associatedprotein 4 Hs.1384 MGMT o-6-methylguanine-DNA methyltransferase Hs.79572Cathepsin D cathepsin D (lysosomal aspartyl protease) Hs.111301Collagenase IV Hs.151738 Collagenase IV Hs.51233 DR5 Hs.82359 FASHs.80409 GADD45 DNA-damage-inducible transcript 1 Hs.86161 GMLGPI-anchored molecule like protein Hs.50649 PIG3 quinone oxidoreductasehomolog Hs.184081 Siah seven in absentia (Drosophila) homolog 1 Hs.56066bFGF fibroblast growth factor 2 (basic) Hs.205902 IGF1-R Hs.21330 MDR1 Pglycoprotein 1/multiple drug resistance 1 Hs.74427 PIG11 Homo sapiensPig11 (PIG11) mRNA, complete cds Hs.76507 PIG7 LPS-induced TNF-alphafactor Hs.8141 PIG8 Hs.146688 PIG12 Hs.104925 PIG10 Hs.202673 PIG6Hs.80642 STAT4 Hs.72988 STAT2 Hs.167503 STAT5A Hs.738 early growthresponse 1 Hs.85148 villin2 Hs.109012 MAD Hs.75251 DEAD/H box bindingprotein 1 Hs.181015 STAT6 Hs.199791 SSI-3 STAT induced STAT inhibitor 3Hs.21486 STAT1 Hs.142258 STAT3 Hs.76578 PIAS3 Protein inhibitor ofactivated STAT3 Hs.44439 CIS4 STAT induced STAT inhibitor 4 Hs.50640SSI-1 JAK binding protein Hs.54483 NMI N-Myc and STAT interactorHs.105779 PIASy Protein inhibitor of activated STAT Hs.110776 STATI2STAT induced STAT inhibitor 2 Hs.181112 EST similar to STAT5A

TABLE 9 Functional Categories of ARGs Tag T/C Access # Name, DescriptionTranscription Regulators GCCAGCCCAG (SEQ ID NO: 13) 11/1  H41030KAP1/TIF1beta, KRAB-associated protein 1 GTGCAGGGAG (SEQ ID NO: 14)18/2  AF071538 PDEF, ets transcription factor GACAAACATT (SEQ ID NO: 15)8/1 NM_003201 mtTF1, mitochondrial transcription factor 1 ATGACTCAAG(SEQ ID NO: 16) 8/1 X12794 ear-2, v-erbA related GAAAAGAAGG (SEQ ID NO:17) 8/1 U80669 Nkx3.1, homeobox CCTGTACCCC (SEQ ID NO: 18) 5/1 AF072836Sox-like transcriptional factor CCTGAACTGG (SEQ ID NO: 19) 1/8 NM_001273CHD4/Mi2-beta, histone acetylase/deacetylase, chromodomain helicaseTGACAGCCCA (SEQ ID NO: 20) 1/7 U81599 Hox B13, homeobox RNA Processingand Translational Regulators TACAAAACCA (SEQ ID NO: 21) 12/1  NM_005381NCL, Nucleolin AATTCTCCTA (SEQ ID NO: 22) 8/1 U41387 GURDB, nucleolarRNA helicase TGCATATCAT (SEQ ID NO: 23) 8/1 D89729 XPO1, exportin 1CTTGACACAC (SEQ ID NO: 24) 14/2  AL080102 EIF5, translation initiationfactor 5 TGTCTAACTA (SEQ ID NO: 25) 5/1 AF078865 CGI-79, RNA-bindingprotein GTGGACCCCA (SEQ ID NO: 26) 10/2  AF190744 SiahBP1/PUF60, poly-Ubinding splicing factor ATAAAGTAAC (SEQ ID NO: 27)  1/11 NM_007178UNRIP, unr-interacting protein. TACATTTTCA (SEQ ID NO: 28) 1/7 X85373SNRPG, small nuclear RNP polypeptide G TCAGAACAGT (SEQ ID NO: 29) 1/7NM_002092 GRSF-1, G-rich RNA binding factor 1 CAACTTCAAC (SEQ ID NO: 30)0/5 NM_006451 PAIP1, poly A BP-interacting protein 1 GATAGGTCGG (SEQ IDNO: 31) 0/5 Z11559 IREBP1, Iron-responsive element BP 1 CTAAAAGGAG (SEQID NO: 32)  2/10 M15919 SNRPE, small nuclear RNP polypeptide E GenomicMaintenance and Cell Cycle Regulation GTGGTGCGTG (SEQ ID NO: 33) 10/1 AF035587 XRCC2, X-ray repair protein 2 TCCCCGTGGC (SEQ ID NO: 34) 7/1D13643 KIAA0018, Dimunuto-like ATTGATCTTG (SEQ ID NO: 35) 6/1 NM_002947RPA3, Replication protein A 14kDa subunit AGCTGGTTTC (SEQ ID NO: 36)16/3  NM_004879 PIG8, p53 induced protein CCTCCCCCGT (SEQ ID NO: 37)10/2  AF044773 BAF, barrier-to-autointegration factor ATGTACTCTG (SEQ IDNO: 38) 1/7 NM_000884 IMPDH2, IMP dehydrogenase 2 GATCAAATAC (SEQ ID NO:39) 0/5 NM_006325 ARA24, androgen receptor assoc protein 24 GTGCATCCCG(SEQ ID NO: 40) 0/5 X16312 Phosvitin/casein kinase II beta subunitProtein Trafficking and Chaperoning GAAATTAGGG (SEQ ID NO: 41) 12/1 AB020637 KIAA0830, similar to golgi antigen TTTCTAGGGG (SEQ ID NO: 42)10/1  AF15189 CGI-140, lysosomal alpha B mannosidase CCCAGGGAGA (SEQ IDNO: 43) 7/1 AF026291 CCT, chaperonin t-complex polypeptide 1 GTGGCGCACA(SEQ ID NO: 44) 13/2  S79862 26 S protease subunit 5b TTGCTTTTGT (SEQ IDNO: 45) 15/3  NM_001660 ARF4, ADP-ribosylation factor 4 ATGTCCTTTC (SEQID NO: 46) 10/2  NM_005570 LMAN1, mannose BP involved in EPR/Golgitraffic Energy Metabolism, Apoptosis and Redox Regulators TGTTTATCCT(SEQ ID NO: 47) 13/2  M14200 DBI, diazepam binding inhibitor GCTTTGTATC(SEQ ID NO: 48) 6/1 D16373 dihydrolipoamide succinyltransferaseGTTCCAGTGA (SEQ ID NO: 49) 6/1 AA653318 FKBP5, FK506-binding protein 5TAGCAGAGGC (SEQ ID NO: 50) 6/1 AA425929 NDUFB10, NADH dehydrogenase 1beta subcomplex 10 ACAAATTATG (SEQ ID NO: 51) 5/1 NM_003375 VDAC,voltage-dependent anion channel CAGTTTGTAC (SEQ ID NO: 52) 5/1 NM_000284PDHA1, Pyruvate dehydrogenase E1-alpha subunit GATTACTTGC (SEQ ID NO:53) 5/1 NM_004813 PEX16, peroxisomal membrane biogenesis factorGGCCAGCCCT (SEQ ID NO: 54) 5/1 X15573 PFKL, 1-phosphofructokinaseCAATTGTAAA (SEQ ID NO: 55)  1/10 NM_004786 TXNL, thioredoxin-likeprotein AAAGCCAAGA (SEQ ID NO: 56)  2/15 NM_001985 ETFB, electrontransfer flavoprotein beta subunit CAACTAATTC (SEQ ID NO: 57) 1/7NM_001831 CLU, Clustrin AAGAGCTAAT (SEQ ID NO: 58) 0/5 NM_004446 EPRS,glutamyl-prolyl-tRNA synthetase Signal Transduction CTTTTCAAGA (SEQ IDNO: 59) 9/1 X59408 CD46, complement system membrane cofactor GTGTGTAAAA(SEQ ID NO: 60) 9/1 NM_005745 BAP31/BAP29 IgD accessory proteinsACAAAATGTA (SEQ ID NO: 61) 8/1 NM_000856 GUCY1A3, Guanylate cyclase 1,alpha 3 AAGGTAGCAG (SEQ ID NO: 62) 7/1 NM_006367 CAP, Adenylylcyclase-associated protein GGCGGGGCCA (SEQ ID NO: 63) 7/1 AB002301microtubule assoc. serine/threonine kinase GGCCAGTAAC (SEQ ID NO: 64)6/1 AL096857 similar to BAT2, integrin receptor AACTTAAGAG (SEQ ID NO:65) 12/2  AB018330 calmodulin-dependent protein kinase kinase βAGGGATGGCC (SEQ ID NO: 66) 5/1 NM_006858 IL1RL1LG, Putative T1/ST2receptor CTTAAGGATT (SEQ ID NO: 67)  2/10 AF151813 CGI-55 protein

The “tag to gene” identification is based on the analysis performed bySAGE software and/or “tag to gene” application of the NIH SAGE Website.T/C represent the number of tags for each transcript in androgen treated(T) and control (C) LNCaP libraries. The differences in expressionlevels of genes identified by tags shown here were statisticallysignificant (p<0.05) as determined by the SAGE software.

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1. A polypeptide, wherein the polypeptide comprises an amino acidsequence that is at least 95% identical to SEQ ID NO:3 and wherein thepolypepide inhibits the growth of LNCaP cells in a colony-forming assay.2. A polypeptide variant of SEQ ID NO:3, wherein the variant comprisesat least one mutation and/or deletion in at least one of the PY motifsof SEQ ID NO:3.
 3. An isolated nucleic acid, wherein the nucleic acidhybridizes to a DNA having the nucleotide sequence of SEQ ID NO:2 underconditions of high stringency, wherein the nucleic acid encodes apolypeptide that inhibits the growth of LNCaP cells in a colony-formingassay.
 4. An isolated antibody that binds to the polypeptide of claim 1.5. An isolated antibody that binds to the polypeptide of claim
 2. 6. Anisolated antibody that binds to the polypeptide of claim
 3. 7. A methodof reducing the expression of an androgen receptor in a prostate cancercell comprising administering a polypeptide according to claim 1 to theprostate cancer cell in an amount effective to reduce expression of theandrogen receptor in the cell.
 8. A method of inhibiting the growth of aprostate cancer cell, comprising administering a polypeptide accordingto claim 1 to the prostate cancer cell in an amount effective to inhibitthe growth of the cancer cell.
 9. A method of modulating the expressionof a gene in a prostate cancer cell, wherein transcription of the geneis regulated by an androgen receptor, comprising administering apolypeptide according to claim 1 to the prostate cancer cell in anamount effective to modulate the expression of the gene in the cell.